bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 004: Plasma Zonulin, Along With a Unique Gut Microbiome Profile, Are Potential Predictors of Systolic Blood Pressure in Humans
Authors: Kim Seungbum , Goel Ruby , Qi Yanfei , Richards Elaine M , Mohammed Mohammed , Handberg Eileen M , Pepine Carl J , Raizada Mohan K
Published: July 2021

Objectives:
We have previously shown that gut pathophysiology and dysbiosis are closely associated with hypertension (HTN) in animal models. However, whether this association also occurs in human HTN is unknown. To address this knowledge gap, we tested two hypotheses. 1. Hypertensive patients (HTN) have gut barrier dysfunction. 2. Prediction of blood pressure will be possible from circulating markers of gut health and gut microbiome composition.

Design and Method:
Plasma and fecal samples were collected from HTN (n=22, mean SBP 155.8±3.4mmHg) and reference subjects without HTN (REF) (n=18, mean SBP 121.1±1.5mmHg) (see ClinicalTrials.gov, NCT02188381 for protocol). Gut microbiomes were analyzed using shotgun metagenomic sequencing and Qiime. Plasma analytes were measured by ELISA.

Results:
Plasma intestinal fatty acid binding protein (REF;1.2 ± 0.1 ng/ml, HTN;1.9 ± 0.2 ng/ml, p=0.0097) and lipopolysaccharide (REF; 39.0 ± 9.5 pg/ml, HTN; 98.0 ± 26.2 pg/ml, p=0.0423) were increased in HTN, suggesting increased intestinal inflammation and permeability. Additionally, the soluble form of zonulin (regulator of gut tight junction proteins) was markedly elevated in the plasma of HTN (REF; 28.4 ± 2.0 ng/ml, HTN; 42.6 ± 2.7 ng/ml, P=0.0002) further supporting gut barrier dysfunction. Plasma zonulin was correlated with SBP (R
2
=0.5301, p<0.0001). Two models predicting SBP were built using stepwise linear regression analysis of microbiome shotgun metagenomics data and circulating markers of gut health. The first model used plasma zonulin as a single predictor, and the second zonulin plus butyrate producing bacteria (adjusted R
2
values of 0.506 and 0.554 respectively, p<0.001 for both). Our first model was validated by prediction of SBP in a separate validation cohort (n=36) from zonulin plasma levels (R
2
=0.4608, p<0.0001).

Conclusions:
Markers of increased gut permeability, particularly zonulin, and abundance of butyrate producing bacteria predicted SBP. These results support the hypothesis that gut barrier dysfunction and gut microbiome composition are directly linked with HTN in humans.
Abstract 007: Obstructive Sleep Apnea Induced Hypertension Involves Gut Dysbiosis and Neuroinflammation
Authors: Durgan David J , Ganesh Bhanu P , Nelson James W , Bryan Robert M
Published: July 2021

Obstructive sleep apnea (OSA) is an independent risk factor for systemic hypertension, and the most common underlying cause of resistant hypertension. The importance of a healthy gut microbiota on host physiology is becoming increasingly evident. We have shown that gut dysbiosis plays a causal role in the development of OSA-induced hypertension. The mechanisms linking gut dysbiosis to hypertension are unknown. We tested the hypothesis that OSA-induced dysbiosis leads to gut barrier dysfunction, systemic inflammation, and neuroinflammation, which is linked to hypertension. We exposed rats to 2 weeks of sham or OSA (60 apneas/hr). OSA led to a >100-fold increase in TNFα expression in the cecum wall (n=5, p<0.001), and decreased goblet cells/crypt (8.4 vs 11.4; n=6, p<0.05). Consistent with gut barrier dysfunction and bacterial translocation, we found bacterial 16S rRNA in adipose tissue, as well as a 4-fold increase in adipose IL-6 mRNA expression following OSA (n=4-7, p<0.05). Flow cytometric analysis revealed a decrease in the percentage of T-reg cells in the brain of OSA vs. sham rats (0.08% vs. 0.25%; n=3, p<0.05). In addition, the percentage of activated microglia was increased following OSA (20% vs. 10%; n=3, p<0.05). Next, we treated sham and OSA rats with a prebiotic (20% resistant starch diet) or probiotic (
C. butyricum
; 10
9
CFU gavage every three days) to increase short chain fatty acids, important in maintaining gut barrier integrity and regulating immune responses. Pre- and probiotic prevented OSA-induced loss of goblet cells and TNFα expression in the cecum. Compared to control rats, pre- and probiotic increased the percentage of T-reg cells in the brain of OSA rats by 10- and 5-fold, respectively (n=3-6, p<0.05 for each). Additionally, pre- and probiotic prevented OSA-induced activation of microglia (n=3-6). Importantly, pre- and probiotic prevented OSA-induced hypertension (prebiotic sham=158.5 vs. OSA=160.3 mmHg, probiotic sham=147.4 vs. OSA=145.6 mmHg; n=6-7, NS). These data demonstrate a causal role for gut dysbiosis in the development of hypertension that involves gut barrier disruption, bacterial translocation, and neuroinflammation. Manipulation of the gut microbiota may serve as a novel therapy in the prevention of hypertension.
Abstract 009: Mitochondria-targeting Overexpression of AT
2
Receptors Inhibits Intracellular Angiotensin II-induced Respiratory and Glycolytic Stress Responses in Mouse Proximal Tubule Cells
Authors: Li Xiao C , Zhuo Jia L
Published: July 2021

Angiotensin II (ANG II) plays an important role in mitochondrial dysfunction associated with cardiovascular, hypertensive and kidney diseases, but it is not known whether extracellular or intracellular ANG II mediates this effect via activation of cell surface or mitochondrial receptors. As a proof of concept study, we overexpressed an intracellular cyan fluorescent ANG II fusion protein, mito-ANG II, with or without a GFP-tagged full length AT
2
receptor, mito-AT
2
R, selectively in the mitochondria of mouse proximal tubule (mPCT) cells. The mitochondrial respiratory and glycolytic stress responses were measured using Seahorse XF Cell Mito and XF Glycolysis Stress Test Kits, respectively. Live cell fluorescent imaging confirmed the expression and colocalization of mito-ANG II and mito-AT
2
R with a mitochondrial marker MitoTracker®. Overexpression of mito-ANG II for 48 h significantly increased mitochondrial oxygen consumption rate (OCR) by 30% (Control: 239.4 ± 9.2 vs. mito-ANG II: 310.4 ± 12.6 pmol/min;
p
<0.01, n=5) and extracellular acidification rate (ECAR) by 33% (Control: 6.3 ± 0.3 vs. mito-ANG II: 8.1 ± 0.5 mpH/min;
p
<0.01, n=5). The effects of mito-ANG II on OCR and ECAR responses were associated with significant increases in phosphorylated MAP kinase ERK1/2, Na
+
/K
+
-ATPase, and mitochondrial redox carries, Complex I (NADH coenzyme Q reductase), Complex II (succinate dehydrogenase), Complex III (cytochrome

bc
1

complex) and Complex IV (cytochrome c oxidase) (
p
<0.01, n=6). The mito-ANG II-induced OCR and ECAR responses were blocked by the AT
1
blocker losartan (10 μM,
p
<0.01, n=5), but not by the AT
2
receptor blocker PD123319 (10 μM,
n.s.
, n=5). However, concurrent overexpression of mito-AT
2
R with mito-ANG II in the mitochondria of mPCT cells significantly attenuated the effects of mito-ANG II on OCR and ECAR responses (
p
<0.01, n=6), while the effects of mito-AT
2
R overexpression were completely blocked by PD123319 (
p
<0.01, n=5). Taken together, our results provide strong evidence that activation of mitochondrial AT
1
receptors by intracellular ANG II stimulates, whereas activation of mitochondrial AT
2
receptors by intracellular ANG II inhibits, mitochondrial respiratory and glycolytic responses in mouse proximal tubule cells.
Abstract 010: Renal Proximal Tubule-derived Angiotensin Converting Enzyme 2 (ACE2) in Blood Pressure Regulation
Authors: Xiang Zhidan , Bendt Katharine , Griffiths Robert , Giani Jorge F , Ortiz Melo David I , Gurley Susan B
Published: July 2021

Angiotensin Converting Enzyme 2 (ACE2) is a carboxypeptidase that metabolizes angiotensin II (AngII) to Ang1-7, playing a critical role in blood pressure (BP) regulation. Previous studies from our lab demonstrated that loss of ACE2 resulted in enhanced susceptibility to AngII hypertension and this was associated with elevated renal AngII levels. ACE2 is highly expressed in kidney, particularly in proximal tubule epithelia, and kidney cross-transplant experiments performed by our group revealed a novel mechanism by which ACE2 may be functioning to regulate BP. However, the exact cellular sources and sites of action where ACE2 regulates BP remain unclear. The objective of this study is to assess proximal tubule-derived ACE2 in BP regulation by studying a new mouse model generated in our laboratory: proximal tubule-specific ACE2 knock-out mice (referred to as ACE2 PTKO mice). ACE2 PTKO mice were generated by crossing the
Ace2
-flox/flox
conditional mouse line with the phosphoenolpyruvate carboxykinase (PEPCK)-Cre mouse (V. Haase, Vanderbilt). ACE2 PTKO mice and their littermate control mice were inbred 129/SvEv, co-caged, and only males were studied. ACE2 PTKO mice exhibited reduced ACE2 mRNA and protein in renal cortex compared to controls (
Ace2
mRNA relative expression: 0.47 ± 0.1 vs. 1.08 ± 0.1, N=3 vs 3,
P
<0.05; relative ACE2 protein expression: 0.38 ± 0.04 vs. 1.15 ± 0.12, N=3 vs 3,
P
<0.01). Measured with radiotelemetry, there were no differences in BPs between ACE2 PTKO mice and control mice (119 ± 1 vs 117 ± 2 mmHg, N=16 vs 16, p=NS) at baseline. However, ACE2 PTKO mice exhibited an enhanced hypertensive response during the first week of AngII infusion compared to control mice (139.9 ± 4.8 vs 152.8 ± 3.7 mmHg at day 7, N=16 vs 16,
P
=0.038). Furthermore, the ACE2 KO kidneys accumulated significantly higher renal AngII than control kidneys, measured at day4 (897±104 vs 536±115 fmol/g,
P
<0.05). Our data demonstrate that proximal tubule-derived ACE2 plays a critical role in BP regulation during AngII infusion. The enhanced hypertensive response in ACE2KO mice is consistent with their intra-renal AngII levels, suggesting that that proximal tubule-derived ACE2 regulates BP via metabolizing AngII within the kidney.
Abstract 018: Neuronal (Pro)renin Receptor Deletion Prevents High-fat Diet Induced High Blood Pressure and Type II Diabetes
Authors: Worker Caleb J , Li Wencheng , Feng Yumei
Published: July 2021

We recently reported that the (pro)renin receptor (PRR) is a key component of the brain renin-angiotensin system, mediating the majority of Ang II formation, and plays a pivotal role in the development of hypertension. Its importance in obesity-related metabolic syndrome is, however, unknown. We hypothesize that brain PRR plays a regulatory role in high-fat diet (HFD) induced metabolic syndrome. To test our hypothesis, neuron-specific PRR knockout (PRRKO) mice and wildtype (WT) littermates were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) with matching calories for 16 weeks. Weekly body weight (BW) and monthly fasting blood glucose (FBG) measurements were recorded and end point glucose tolerance (GTT) and insulin sensitivity tests (IST) were performed. Blood pressure (BP) was recorded using radiotelemetry in conscious free moving mice. We observed no difference in BW or food intake between genotypes in either HFD or NFD. The baseline BP and heart rate (HR) were similar between PRRKO and WT mice; however, following 16 weeks HFD the BP (101±6 vs. 111±3 mmHg, P=0.035) and HR (536±12 vs. 578±4 BPM, P=0.046) were significantly lower in PRRKO compared with WT mice. Interestingly, neuronal PRR deletion attenuated the elevation of FBG (127.12±10.46 vs. 167.77±16.57 mg/dl, P=0.039) induced by HFD. Glucose tolerance was significantly improved in PRRKO compared with WT following 16 weeks of HFD (AUC: 20557±894 vs. 29994±2976, P=0.006), while there was no difference in the IST between the groups. We also found that HFD mice had higher levels of plasma (pro)renin (9.95±1.83 vs. 2.74± 0.47 ng/ml, P=0.005) and brain angiotensin II (656.8±94.9 vs. 375.3±32.0 pg/g, P=0.02), as well as higher cardiac (ΔHR to propranolol: -150±6 vs. -82±15 bpm , P=0.0054) and vasomotor (ΔBP to chlorisondamine: -44±3 vs. -22±3 mmHg, P=0.0004) sympathetic tone, suggesting that the HFD-induced rise in BP is sympathetically mediated and associated with elevation of brain angiotensin II. Our data indicates that PRR deletion in the neurons protects against glucose intolerance and BP elevation in HFD mice with no effect on insulin sensitivity or body weight. We conclude that neuronal PRR plays a role in the development of obesity-related metabolic syndrome.
Abstract 027: ProANP 31-67 and Renal Function in Heart Failure
Authors: Felder Robin , Schiermeyer Katie , Xu Peng , Gildea John J , Geimer T R , Mahan Larry
Published: July 2021

The physiologic interplay between heart and kidney is best understood in the context of heart failure (HF). Two-thirds of HF patients suffer from cardiorenal syndrome (CRS) - heart failure with renal insufficiency wherein both organs contribute to their reciprocal decline. Four peptides are released from an atrial natriuretic prohormone in response to atrial-ventricular wall stress in HF. One in particular, a well characterized 28 a.a cyclic peptide from the C-terminus termed ANP elicits systemic vasodilation via cGMP release and was developed as Natrecor® to treat acute HF. Limited preclinical data exists regarding the remaining three N-terminal peptides although all three have been explored for clinical utility in a limited series of Phase 1 and 2a clinical trials in the U.S. and in Australia. We have examined a 37 a.a. linear peptide, mid-sequence in the prohormone termed proANP 31-67 (MP3167) to better understand its role in CRS. MP3167 is unique among the four as it has the longest half-life of all in humans (1-1/2 hours vs. minutes), is highly acidic (pI=3.2; -5 net charge), appears resistant to degradation by neprilysin and has been reported to act through PGE2 although the tissue source in humans is unknown. We show for the first time that MP3167 elicits a 3-fold increase in PGE2 release over basal levels predominantly in
human renal medullary tissue
(from 2401 ± 97.2 to 8437 ± 62.9 pg/ml, N=3, p<0.001). The positive control, beta hydroxyl butyric acid (BOH) also increased PGe2 (from 2401 ± 97.2 to 7749 ±120 pg/ml, N=3, p<0.01). In
3D cultured cells isolated from digested tissue
, we also observed an increase in MP3167 stimulated PGE2 (From 41.1 ± 1.3 to 62.9 ± 3.19 pg/ml, N=3, p<0.05). The PGE2 signal co-localizes with cells stained by L1-CAM antibody implicating collecting tubules/ducts at the cortical-medullary interface. As the receptor and second messenger pathway for MP3167 is unknown, we have isolated L1-CAM positive cells and created immortalized cell lines of human origin. Initial findings reiterate MP3167 stimulated PGE2 release, reaching a maximum by ~10 min. These human studies will enable better understanding of MP3167 as a regulator of renal function and the paracrine actions of PGE2 in rebalancing heart and renal function in CRS associated HF.
Abstract 028: Endothelial Nogo-B Controls Sphingolipid de novo Biosynthesis to Impact Coronary Artery Atherosclerosis
Authors: Zhang Yi , Sasset Linda , Xu Ren , Blake Greenblatt Matthew , Di Lorenzo Annarita
Published: July 2021

Coronary artery disease is a leading cause of myocardial infarction (MI) worldwide. Alterations in sphingolipid levels have been linked to atherosclerosis although specific molecular mechanisms are poorly understood. Recently, we discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular functions by inhibiting serine palmitoyltransferase (SPT), the rate-limiting enzyme of the
de novo
sphingolipid biosynthesis. Mice lacking Nogo-B are resistant to hypertension and heart failure. Here, we employed a novel model of coronary atherosclerotic lesions induced by hypercholesterolemia and hypertension, well-known risk factors for atherosclerosis. To this aim, transverse aortic constriction (TAC) surgery was performed in mice lacking endothelial Nogo-B in ApoE
-/-
background and ApoE
-/-
mice as control. ApoE
-/-
mice developed coronary atherosclerotic lesions within 6 weeks following TAC, (without the need of long-term high-cholesterol diet) and 70% of the mice died of MI at 6-week post-TAC. On the contrary, mice lacking Nogo-B specifically in endothelial cells were markedly resistant to the development of coronary atherosclerotic lesions and MI (20%). Mechanistically, in the absence of endothelial Nogo-B, the biosynthesis of sphingolipids, particularly S1P, is upregulated, protecting the endothelium from hypertension and hypercholesterolemia-triggered vascular inflammation and atherogenesis. This study identifies an important and novel role of endothelial Nogo-B-dependent regulation of sphingolipid
de novo
biosynthesis in the coronary atherosclerosis, a primary cause of myocardial infarction.
Abstract 037: Exaggerated Placental Ischemia-induced Hypertension in Endothelin Receptor Type B (ETB)-deficient Pregnant Rats s Independent of Increased sFlt-1 or ROS Levels
Authors: Spradley Frank T
Published: July 2021

While the pathogenesis of preeclampsia is not fully understood, studies implicate placental ischemia. Reduced uterine perfusion pressure (RUPP)-induced placental ischemia/hypoxia in animal models stimulates release of factors like antiangiogenic sFlt-1 into the maternal circulation increasing vascular-renal ET-1. ET-1 promotes hypertension via reactive oxygen species (ROS). Blockade of vasoconstrictive ETA abolishes RUPP hypertension. Deficiency of vasodilatory ETB in rats leads to increased blood pressure in pregnancy. While ETB deficiency markedly enhances RUPP hypertension, it is unknown if there is exaggerated RUPP-induced sFlt-1, ET-1 or ROS levels in ETB-def rats. The hypothesis was tested that placental ischemia/hypoxia-induced release of sFlt-1 and circulating ET-1 and ROS are greater in ETB-def rats. Eighteen-week-old ETB-def and transgenic (Tg) control pregnant rats were generated with Wistar Hannover males. RUPP or Sham surgeries were on gestational day 14 and assessment of plasmas and placentas at day 19. RUPP increased placental sFlt-1 (pg/mg) similarly in RUPP ETB-def (781±113, N=5) vs Sham ETB-def (573±54, N=12) and RUPP Tg (631±62, N=5) vs Sham Tg (547±31, N=12) (P<0.05). In placental explant cultures, acute hypoxia (48 h 1% O2 vs normoxia 6% O2) stimulated a comparable release of sFlt-1 (pg/mg) in Sham ETB-def (2577±135 vs 2070±78) and Sham Tg (3208±318 vs 2553±107) (P<0.05). Unexpectedly, plasma sFlt-1 (pg/mL) was lower in RUPP ETB-def (153±48) vs Sham ETB-def (476±125) and RUPP Tg (238±32) vs Sham Tg (463±102) (P<0.05). Plasma ET-1 (fmol/L) was exaggerated in RUPP ETB-def (954±70) and greater in Sham ETB-def (735±43) vs RUPP Tg (122±14) or Sham Tg (142±41) (P<0.05). Plasma H2O2 (umol/L) was not exaggerated in RUPP ETB-def (5.4±1.2) or RUPP Tg (4.0±0.5) but was greater (P<0.05) in Sham ETB-def (6.2±0.3) vs Sham Tg (3.6±0.3). In conclusion, these data suggest in 1) normal pregnancy, ETB is crucial for blood pressure control by regulating bioavailable ET-1 to prevent ROS production and 2) placental ischemia, ETB reduces excess ET-1 to buffer hypertension independently of sFlt-1 or ROS. These data support ETB physiology as important in controlling blood pressure in pregnancy and its loss in mediating hypertension in preeclampsia.
Abstract 042: Evidence of Angiotensin II (AngII)-dependent Obesity-induced Hypertension in Female Mice Exposed to Postnatal Neglect
Authors: Mitchell Kierrariel , Cohn Dianne , Loria Analia S
Published: July 2021

Previously, we have shown that female mice subjected to maternal separation with early weaning (MSEW), a model of postnatal neglect, display exacerbated diet-induced obesity and high blood pressure (BP) compared with control mice. Female MSEW mice show activated renin-angiotensin system components, including increased plasma renin activity and adipose tissue-derived angiotensinogen secretion. The goal of this study was to test whether augmented obesity-induced hypertension in female MSEW mice is AngII-dependent. Mouse MSEW was achieved by repeated, daily separations from the dam and 4-day early weaning. Normally reared controls (C) were weaned at postnatal day 21. Each experimental group of female weanlings was comprised of 6 mice each and derived from 3 different litters, that were placed on high fat diet (HFD, 60% kcal from fat). After 18 weeks, mice were implanted with radiotelemetry for BP measurement. At week 20, average 24-hr systolic blood pressure (SBP) was 134±2 mmHg in MSEW mice and 126±2 in C (P<0.05). No significant changes in mean arterial pressure, diastolic blood pressure or heart rate were observed between groups. Next, we also determined the BP sensitivity to the acute administration of AngII (1, 10 and 50 ug/kg, s.c.). AngII-induced BP changes, assessed by BP area under the curve, were similar between MSEW and C mice at all doses (50 ug/kg dose:145±10 vs. 132±15 mmHgx30 min, respectively). Chronic enalapril treatment (2.5 mg/kg/day, drinking water, 7 days) was conducted to block endogenous AngII synthesis. Enalapril reduced SBP 15±2 mmHg in MSEW mice but only 6±1 mmHg in C mice (p<0.05). The BP response to acute AngII doses increased similarly in MSEW and C enalapril-treated mice, (50 ug/kg dose: 200±13 vs. 207±22 mmHgx30 min, respectively). In addition, BP and HR responses to acute injections (i.p) of mecamylamine (5 mg/kg), propranolol (5 mg/kg) or atropine (1 mg/kg) were similar between untreated MSEW and C mice, suggesting that exacerbated BP in female MSEW mice is independent of sympathetic or parasympathetic dysfunction. Taken together, these data provide evidence that increased BP in female MSEW mice results from elevated circulating AngII rather than enhanced AngII sensitivity or sympathetic nerve activity.
Abstract 051: Influence of Sex and Obesity on the Effect of Preterm Birth on the Renin-angiotensin System in Adolescents
Authors: South Andrew M , Nixon Patricia A , Chappell Mark C , Diz Debra I , Russell Gregory B , Jensen Elizabeth T , Shaltout Hossam A , Washburn Lisa K
Published: July 2021

Background:
Preterm birth increases the risk of cardiovascular disease, but the underlying mechanisms are not known. Prematurity may induce programming effects that differentially influence the renin-angiotensin (Ang) system (RAS), particularly suppression of the beneficial Ang-(1-7) axis. Patient factors such as sex and obesity may influence the degree of RAS programming. Therefore, we hypothesize that preterm birth is associated with alterations in the RAS in adolescence in a sex and adiposity-dependent manner.

Methods:
We evaluated a cohort of 175 adolescents born preterm and 51 term controls at age 14 years. We recorded systolic and diastolic BP z-scores, measured Ang II and Ang-(1-7) levels in plasma and urine, and calculated the peptide ratios. We applied generalized linear models to estimate the association between preterm birth and the RAS, adjusting for race, socioeconomic status, and maternal hypertension and smoking; the models were stratified by sex and overweight/obesity (body mass index ≥85
th
%ile for age and sex).

Results:
Mean systolic and diastolic BP z-scores were higher among those born preterm (
p
<0.001 and
p
=0.03, respectively). Relative to term birth, preterm birth was associated with an increased plasma ratio of Ang II to Ang-(1-7) (
β
: 4.42, 95% CI 1.52 to 7.32), decreased Ang II (
β
: -5.16 pmol/L, -10.28 to -0.04), decreased Ang-(1-7) (
β
: -5.38 pmol/L, -8.66 to -2.09), and a decreased urinary ratio of Ang II to Ang-(1-7) (
β
: -0.13, -0.26 to -0.003). In stratified analyses, female sex (
β
: -7.14 pmol/L, -11.03 to -3.24) and overweight/obesity (
β
: -8.21 pmol/L, -12.51 to -3.91) were associated with greater reductions in plasma Ang-(1-7). Overweight/obesity was associated with a greater increase in the ratio of plasma Ang II to Ang-(1-7) (
β
: +6.13, 0.58 to 11.68).

Conclusions:
Circulating Ang-(1-7) was lower relative to Ang II in adolescents born preterm. This suggests fetal RAS programming may contribute to the increased risk of cardiovascular disease in those born preterm. We note an important influence of sex in that the decrease in Ang-(1-7) is intensified in girls. Moreover, obesity may confer a second physiologic insult through a higher Ang II/Ang-(1-7) that exacerbates the risk of cardiovascular disease, including hypertension.
Abstract 056: Differential Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Suggest GRK5-Mediated Cardio-protection Against Mineralocorticoids
Authors: Lymperopoulos Anastasios , McCrink Katie A , Brill Ava , Maning Jennifer , Desimine Victoria L , Aukszi Beatrix
Published: July 2021

Background:
Aldosterone (Aldo) contributes significantly to the morbidity & mortality of heart failure (HF). In the heart, Aldo binds the mineralocorticoid receptor (MR) to exert damaging effects, e.g. fibrosis, apoptosis, oxidative stress, etc. Additionally, Aldo can activate the G protein-coupled estrogen receptor (GPER), which may have beneficial, anti-apoptotic effects for cardiomyocytes, e.g. in ischemia/reperfusion injury. GRK2 and GRK5 are the most abundant G protein-coupled receptor (GPCR)-kinases (GRKs) in the heart and both phosphorylate GPCRs but also non-GPCR substrates. The human MR is known to undergo inhibitory phosphorylation at Ser-843, inside its ligand binding domain, which blocks its transcriptional activity.

Hypothesis:
In the heart, GRK5 phosphorylates and inhibits the MR, whereas GRK2 phosphorylates and desensitizes GPER.

Methods:
We used the cardiomyocyte cell line H9c2 and isolated adult rat ventricular myocytes (ARVMs). We performed co-immunoprecipitation experiments for GRK interactions with MR or GPER. We measured MR phosphorylation via immunoblotting and MR transcriptional activity via the luciferase reporter assay. We also measured apoptosis and oxidative stress in ARVMs.

Results:
GRK5, but not GRK2, phosphorylates the MR in H9c2 cardiomyocytes. Beta
2
-adrenoceptor activation stimulates this non-canonical effect of GRK5. In contrast, GRK2, but not GRK5, phosphorylates and desensitizes agonist-activated GPER. The GRK5-phosphorylated MR is incapable of activating gene transcription, since MR transcriptional activity is markedly suppressed upon GRK5 overexpression. Conversely, CRISPR-mediated GRK5 gene deletion augments cardiac MR transcriptional activity. Importantly, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis/oxidative stress in ARVMs. Finally, beta
2
-adrenoceptor-stimulated GRK5 phosphorylates and inhibits the MR in ARVMs.

Conclusions:
GRK5 blocks the cardio-toxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder GPER`s cardio-protective effects. Thus, cardiac GRK5 stimulation (e.g. via beta
2
-adrenoceptor activation) enhances the cardio-protection exerted by Aldo inhibitors in HF.
Abstract 057: Cellular and Genetic Causes of Idiopathic Hyperaldosteronism
Authors: Omata Kei , Satoh Fumitoshi , Morimoto Ryo , Ito Sadayoshi , Yamazaki Yuto , Nakamura Yasuhiro , Anand Sharath , Guo Zeng , Stowasser Michael , Sasano Hironobu , Tomlins Scott , Rainey William
Published: July 2021

Background:
Primary aldosteronism (PA) affects ~10% of hypertensive patients and has unilateral and bilateral forms (30%:70%). Unilateral PA is caused by aldosterone-producing adenomas (APA), which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone regulating genes (
KCNJ5>>CACNA1D
). We recently demonstrated that adrenals from normotensive patients present with pockets of cell expressing aldosterone synthase (CYP11B2). These aldosterone-producing foci (APF) have somatic gene mutations similar to those found in APA (
CACNA1D
>>
KCNJ5
). Bilateral PA, which is typically treated by mineralocorticoid receptor (MR) blockade rather than surgery, is termed idiopathic hyperaldosteronism (IHA). Its pathobiology is largely unknown but has been thought to be due to zona glomerulosa (ZG) hyperplasia.

Methods:
We studied 11 IHA patients (7 males, 4 females) who had unilateral adrenalectomy. Immunohistochemistry for CYP11B2 and next generation sequencing (NGS) targeting genes found in APA were performed on formalin fixed paraffin embedded adrenal tissue. Results were compared to previously described cohorts of 53 age-matched normotensive patients (29 males, 24 females) which were evaluated similarly.

Results:
CYP11B2 expression was absent from intervening ZG cells in 8/11 (73%) IHA adrenals, but all adrenals harbored at least one APF. The median number and size of APF per case were significantly larger in IHA than normotensive controls (6.1 vs 0 APF/cm
2

of adrenal cortex and 0.25 vs. 0.16 mm
2
, respectively; p<0.0001 and p<0.006). In this IHA cohort, NGS identified
CACNA1D
and
KCNJ5
somatic mutations in 44/71 (62%) and 1/71 (1%) of APF, respectively.
Interpretations.
Diffuse CYP11B2 expression in adrenal ZG cells was only observed in 3/11 IHA cases, arguing against ZG hyperplasia as the major underlying pathobiology. Rather, we demonstrated increased and enlarged APF in IHA adrenals compared to normotensive controls, supporting potential contribution to the clinical manifestations of hyperaldosteronism. The frequent occurrence of aldosterone-dysregulating
CACNA1D
somatic mutations in APF support
CACNA1D
as a potential therapeutic target in IHA to complement current MR blockade approaches.
Abstract 064: Transactivation of Egfr by Tgfβ Induces Hypertension by Increasing Arteriolar Myogenic Response
Authors: Carnevale Daniela , Casaburo Manuel , Fardella Stefania , Cifelli Giuseppe , Innocenzi Gualtiero , Bressan Giorgio , Lembo Giuseppe
Published: July 2021

Emilin1 (E1) is a protein of the extracellular matrix regulating TGFβ bioavailability through proTGFβ proteolysis. E1 KO mice are hypertensive with increased TGFβ activation. As E1 is expressed in vessels from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of homeostatic role in the adult. To dissect this issue, we used a conditional gene targeting inactivating E1 in smooth muscle cells (SMCs) of adult mice, by the use of E1flox/flox and tamoxifen (TAM) inducible Cre recombinase specific for SMCs. When E1fl/fl mice carrying the Smmhc-CreERT2 were given TAM blood pressure significantly increased (SBP: 123±2 vs basal condition 104±3;***p< 0.001) as well as myogenic tone (MT) of resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). How increased TGFβ signaling in SMCs could determine an increased MT is still unknown. Relevant to this, we found that the higher TGFβ signaling in E1 KO SMCs stimulates heparin binding epidermal growth factor (HB-EGF) and subsequent transactivation of the EGF receptor, a mechanism typically implied in potentiating MT. When mesenteric resistance arteries from E1 were subjected to step-increases in intraluminal pressure, EGFR inhibition rescued the increased MT. At the molecular level, TGFβ-induced EGFR transactivation resulted into the activation of transient receptor potential classical type 6 (TRPC6) and melastatin type 4 (TRPM4) channels. To put our data into translational perspective, we measured MT of resistance arteries isolated from hypertensive patients and normotensive subjects, finding increased MT (HT 16.4±0.7; NT 11±0.4;***p< 0.001) and TGFβ signaling in the former group. By using a neutralizing anti-TGFβ or an anti-EGFR we found a normalization of the increased MT (HT+anti-TGFβ 11±1.3; NT+anti-TGFβ 11.2±1.2;***p< 0.001 and HT+anti-EGFR 9±0.6; NT+anti-EGFR 10.8±1.1;***p< 0.001), thus confirming the relevance of TGFβ-EGFR pathway in humans. Taken together these data suggest that primary increase of MT induced by TGFβ-EGFR transactivation can cause hypertension and that higher TGFβ signaling and MT are common alterations of resistance arteries of hypertensive patients.
Abstract 083: Neurons in the Organum Vasculosum of the Lamina Terminalis Contribute to Salt-sensitive Hypertension
Authors: Stocker Sean D
Published: July 2021

Excess dietary salt intake raises plasma and cerebrospinal fluid NaCl concentrations to elevate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). Changes in extracellular NaCl concentrations are sensed by neurons in the organum vasculosum of the lamina terminalis (OVLT) - a circumventricular organ that lacks a complete blood-brain barrier. The purpose of the present study was to investigate the hypothesis that salt-sensitive hypertension was mediated, in part, by an elevated activity of OVLT neurons. Dahl-Salt-Sensitive or Sprague-Dawley rats (8-10 weeks) were fed 0.5% or 4.0% NaCl diets for 3-4 weeks. First, in vivo single-unit recordings demonstrate the discharge of OVLT neurons in Dahl-Salt-Sensitive rats was higher after a 4.0% versus 0.5% NaCl diet (4.1±0.4 Hz vs 1.9±0.3 Hz, n=6 per group, P<0.05). OVLT neuronal discharge of Sprague-Dawley rats was not different after a 4.0% or 0.5% NaCl diet (2.1±0.4 Hz vs 1.7±0.3 Hz, n=6-9 per group, P>0.5). In a second set of experiments, injection of hypertonic NaCl (1.0M NaCl, 20nL) into the OVLT produced significantly greater increases in lumbar SNA (131±6% vs 116±3%, n=4 per group, P<0.05) and mean ABP (14±2 vs 8±2 mmHg, n=4 per group, P<0.05) of Dahl-Salt-Sensitive rats fed 4.0% versus 0.5% NaCl respectively. Sprague-Dawley rats fed 4.0% versus 0.5% NaCl exhibited responses of smaller magnitude for both lumbar SNA (115±4 vs 108±3%, n=4 per group, P<0.05) and mean ABP (9±2 vs 6±2 mmHg, n=4 per group, P<0.05). Interestingly, the duration of the response was much longer in Dahl-Salt-Sensitive versus Sprague-Dawley rats (data not shown). Finally, inhibition of neuronal activity by injection of the GABA agonist muscimol (5mM, 20nL) into the OVLT produced a significantly greater fall in lumbar SNA (-25±4% vs -11±3%, n=4 per group, P<0.05) and mean ABP (-19±4 vs -6±2 mmHg, n=4 per group, P<0.05) of Dahl-Salt-Sensitive rats fed 4.0% versus 0.5% NaCl, respectively. Injection of muscimol into the OVLT of Sprague-Dawley rats did not significantly affect SNA or mean ABP. Collectively, these findings suggest a high salt diet increases the activity of OVLT neurons to elevate SNA and ABP in salt-sensitive hypertension.
Abstract 094: Predicting the Risk of Apparent Treatment Resistant Hypertension: A Retrospective, Longitudinal, Cohort Study in an Urban Hypertension Referral Clinic Setting
Authors: Buhnerkempe Michael G , Botchway Albert , Nolasco-Morales Carlos , Prakash Vivek , Hedquist Lowell , Flack John M
Published: July 2021

Background:
Apparent treatment resistant hypertension (aTRH) is associated with increased prevalence of secondary hypertension and adverse pressure-related clinical outcomes. We previously showed that cross-sectional prevalence estimates of aTRH are lower than its true prevalence as patients with uncontrolled hypertension undergoing intensification/optimization of therapy will, over time, increasingly satisfy diagnostic criteria for aTRH.

Methods:
aTRH (SBP and/or DBP at or above a clinically defined goal BP [140/90, 130/85, 130/80, or 125/75 mmHg] over two consecutive office visits when on ≥ 3 antihypertensive drug classes, including a diuretic; or SBP and DBP below goal when on ≥ 4 drug classes, including a diuretic) was assessed in an urban referral hypertension clinic in 924 patients ≥ 30 years old (57.7 ± 12.6) with at least two follow-up visits over 240 days. Patients were mostly African-American (86%; 795/924) and female (65%; 601/924). A minority (28.7%; 265/924) were taking diuretics at their index visit, and analyses were stratified according to this use. Risk for aTRH was estimated using logistic regression with patient characteristics at index visit as predictors. Performance of this risk score at discriminating aTRH status over follow-up was assessed using AUC and was internally validated using bootstrapping.

Results:
Amongst those on diuretics, 80/265 (30.2%) developed aTRH; the risk score discriminated well (AUC = 0.79, bootstrapped 95% CI [0.73, 0.84]). In patients not on a diuretic, 151/659 (22.9%) developed aTRH, and the risk score showed moderate, but significantly lower, discriminative ability (AUC = 0.71 [0.66, 0.74]; p < 0.001). In the diuretic and non-diuretic cohorts, 43/265 (16.2%) and 101/265 (38.1%) of patients, respectively, had estimated risks for development of aTRH < 10%. Of these low-risk patients, 42/43 (97.7%) and 97/101 (96.0%) did not develop aTRH (negative predictive value, diuretics – 0.95 [0.93, 1.00], no diuretics – 0.96 [0.91, 1.00]).

Conclusions:
We created a novel clinical score that discriminates well between those who will and will not develop aTRH, especially amongst those taking diuretics initially. Irrespective of diuretic treatment status, a low risk score had very high negative predictive value.
Abstract 107: Role of Nox4 in the Control of ENaC Activity in Dahl SS Rats During the Development of Salt-induced Hypertension and Diabetic Nephropathy
Authors: Pavlov Tengis S , Ilatovskaya Daria V , Blass Gregory , Palygin Oleg , Levchenko Vladislav , Cowley Allen W , Staruschenko Alexander
Published: July 2021

Dahl salt sensitive (SS) rat is a well-established model for studying salt-induced hypertension and associated kidney injury. We and others have previously shown that salt-sensitive hypertension is accompanied by increased renal production of reactive oxygen species (ROS) and excessive activity of ENaC in the distal nephron. To investigate role of ROS, and specifically NADPH oxidase 4 (Nox4), a primary source of ROS in the kidney, involved in the regulation of ENaC activity during the development of SS hypertension and type 1 diabetes, we performed patch clamp analysis in the cortical collecting ducts of Dahl SS rats and SS rats lacking Nox4 (Nox4
-/-
). We found that SS rats fed a 4% NaCl diet have significantly elevated ENaC activity even 3 days post diet change. ENaC activity (NP
o
) was 0.57±0.08, 1.32±0.3 and 1.69±0.06 before, 3 days and 3 weeks after high salt diet, respectively. In contrast, ENaC activity was not significantly different in SS
Nox4-/-

animals after high salt diet. To study the role of Nox4 in hyperglycemic conditions, diabetes was induced in 6 weeks old male wild type or Nox4
-/-
rats with a single i.p. injection of STZ. We found that ENaC activity in the animals that were hyperglycemic for 11 weeks was elevated compared to control rats (0.71±0.10 and 1.27±0.2; p<0.05) and this effect was mediated via changes in channel open probability. Nox4 deficiency blunts the effect of hyperglycemia on ENaC activity (in STZ-treated animals open probability significantly increased from 0.43±0.06 to 0.86±0.07 in WT rats, but in the Nox4
-/-
group did not change: P
o
was 0.51±0.06 and 0.51±0.07, respectively) that delineates the importance of Nox4-mediated ROS production for regulation of ENaC open probability. Taken together, our data indicate that ENaC activity in Dahl SS rats is elevated following a change of salt diet (3 days and 3 weeks at high salt) and after the development of type 1 diabetes. Furthermore, Nox4 plays a crucial role in these effects of high salt and hyperglycemia on ENaC activity.
Abstract 116: Potent Hypertensive Actions of Angiotensin-sensitive Neurons Within the Lamina Terminalis
Authors: De Kloet Annette D , Domingos-Souza Gean , Smeltzer Michael D , Tan Yalun , Cahill Karlena , Bruce Erin B , Raizada Mohan K , Sumners Colin , Krause Eric G
Published: July 2021

It is accepted that activation of angiotensin type 1a receptors (AT1a) within the CNS elevates blood pressure by influencing sympathetic outflow and vasopressin (VP) secretion; however, the neuronal circuits mediating these effects are not completely understood. The present studies characterize the structure and function of AT1a neurons residing in the median preoptic nucleus (MnPO) and the organum vasculosum of the lamina terminalis (OVLT), thereby evaluating their potential role in blood pressure control. Using male mice that express Cre-recombinase prior to the STOP codon of the AT1a gene, initial studies combined genetic reporting with in situ hybridization to reveal that AT1a neurons in the MnPO and OVLT are largely excitatory (87±4% express vesicular glutamate transporter 2). Subsequently, AT1a-Cre mice were delivered a Cre-inducible adeno-associated virus to induce expression of channelrhodopsin-2 (ChR2) and enhanced yellow fluorescent protein (eYFP) specifically within AT1a neurons of the MnPO/OVLT (AAV-ChR2-eYFP; n = 4). Control mice were delivered AAV-eYFP (n = 4). Analysis of eYFP immunofluorescence revealed that neurons within the MnPO/OVLT that express AT1a send projections to the paraventricular nucleus of the hypothalamus (PVN; an area involved in sympathetic outflow and VP secretion) that appear to synapse onto VP synthesizing neurons. To evaluate the functionality of this connection, we optogenetically stimulated AT1a neurons in the region while recording cardiovascular parameters in anesthetized mice. Ten-minutes of optogenetic stimulation (473 nM;15 Hz; 20 ms pulse width; 60 s ON/OFF) robustly elevated systolic blood pressure in AAV-ChR2-eYFP mice relative to AAV-eYFP controls. This effect was rapid in its onset (34 ± 9 vs. -2 ± 4 mmHg at 5 min, p < 0.05) but persisted for the entire 50 min of cardiovascular recording (45 ± 11 vs. -3 ± 7 mmHg at 50 min, p < 0.05). Intriguingly, the optogenetic stimulation also resulted in 62% increase in Fos induction in AVP neurons within the PVN relative to AAV-eYFP controls (86 ± 5 vs. 52 ± 2 %; p<0.01). Collectively, these results suggest that excitation of AT1a neurons in the MnPO/OVLT recruits autonomic and neuroendocrine responses that promote robust and sustained increases in blood pressure.
Abstract 125: Elevated Muscle Sympathetic Nerve Activity is Associated With Higher Aortic and Carotid Stiffness Independent of Blood Pressure in Women
Authors: Holwerda Seth W , Luehrs Rachel E , Wooldridge Nealy A , DuBose Lyndsey E , Fiedorowicz Jess G , Pierce Gary L
Published: July 2021

Central arterial stiffness, a significant contributor to the development of hypertension and cardiovascular disease with aging, is linked to elevated muscle sympathetic nerve activity (MSNA) in men. However, the extent to which MSNA is associated with central arterial stiffness in women is unknown. Given that the age-related increase in MSNA and arterial blood pressure (BP) occurs at a steeper rate among women compared to men, we tested the hypothesis that resting MSNA is more strongly correlated with central arterial stiffness in women than in men. Also, because of the parallel age-related increase in MSNA, we further hypothesized that the relation between MSNA and central arterial stiffness would not be independent of age. MSNA (microneurography), aortic stiffness (carotid-femoral pulse wave velocity, CFPWV), and carotid β-stiffness (carotid tonometry and ultrasound) were assessed in 54 healthy men (n=29; 19-72 yrs; 30 ± 1 kg/m
2
; systolic BP: 128 ± 3 mmHg) and women (n=26; 26-64 yrs; 29 ± 2 kg/m
2
; systolic BP: 116 ± 3 mmHg). No differences between men and women were observed for CFPWV (Men: 7.0 ± 0.3 vs. Women: 6.8 ± 0.4 mmHg, P=0.747) and carotid β-stiffness (Men: 7.6 ± 0.8 vs. Women: 7.6 ± 0.5 mmHg, P=0.975). Mean BP was lower in women compared to men (Men: 93 ± 3 vs. Women: 85 ± 2 mmHg, P=0.021) and MSNA tended to be lower in women compared to men (Men: 25 ± 3 vs. Women: 20 ± 2 bursts/min, P=0.091). After adjusting for mean BP and HR (partial correlation), CFPWV was significantly correlated with MSNA in men (R=0.44, P=0.021) and women (R=0.58, P=0.004). Interestingly, further adjustment for age abolished the association between CFPWV and MSNA in men (R=0.01, P=0.968), but not in women (R=0.43, P=0.046). A moderate relation between carotid β-stiffness and MSNA was observed in men (R=0.37, P=0.063) and women (R=0.44, P=0.034), but was abolished after adjusting for age (Men: R=-0.001, P=0.995; Women: R=0.26, P=0.245). These preliminary data demonstrate that MSNA is positively correlated with central arterial stiffness in women and men independent of BP. Furthermore, abolishment of the relation between MSNA and CFPWV in men only when adjusting for age suggests that the association between MSNA and central arterial stiffness may be more robust in women.
Abstract 148: Association of Socioeconomic Context With Blood Pressure Response and Cardiovascular Outcomes in ALLHAT
Authors: Shahu Andi , Herrin Jeph , Dhruva Sanket S , Desai Nihar R , Krumholz Harlan M , Spatz Erica S
Published: July 2021

Introduction:
Observational studies demonstrate that in low socioeconomic (SE) communities, where residents face social challenges and fewer opportunities for healthy lifestyle behaviors or quality care, blood pressure (BP) is higher and there are worse cardiovascular (CV) outcomes. Yet whether the effectiveness of antihypertensive therapy on BP control and CV outcomes varies by SE context in a randomized clinical trial (RCT) - where patients are treated under a standard protocol - is unknown.

Methods:
We used data from the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), which randomized 42,418 people with hypertension (HTN) to chlorthalidone, lisinopril, amlodipine or doxazosin. After excluding non-continental US sites and the doxazosin arm (terminated early), our study included 27,862 participants. We defined SE context by mapping study site ZIP codes to counties and stratifying these into income quintiles based on the national distribution of cost-of-living adjusted county median household income (2000 US Census). We compared characteristics and outcomes between participants in the lowest and highest SE quintiles using multivariable regression models. We replicated the analysis with black participants (n = 10532).

Results:
Participants enrolled in low SE sites (n = 2169, 7.8%) were more likely to be female, black, Hispanic, less educated, live in the South, and have fewer CV risk factors than participants in high SE sites (n = 10458, 37.6%). Participants in low SE sites were less likely to achieve BP control (<140/90 mmHg) at 6 years (OR 0.48, 95% CI [0.37, 0.63]). They had higher all-cause mortality (HR 1.25, 95% CI [1.10, 1.41]) and heart failure hospitalization/mortality (HR 1.26, 95% CI [1.03, 1.55]), though lower angina hospitalization (HR 0.70, 95% CI [0.59, 0.83]) and coronary revascularization (HR 0.71, 95% CI [0.57, 0.89]) and no differences in stroke or MI. Results were similar among black participants.

Conclusion:
Despite having access to extra resources afforded by an RCT, ALLHAT participants in low SE sites had poorer BP control, higher mortality and greater heart failure morbidity. Our findings suggest a need to address the SE context beyond the medical environment to attain equity in HTN outcomes.