bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2011: Chronic Systemic Administration of Pro-Inflammatory Cytokines Causes Cardiovascular Dysfunction and Downregulates Brain Expression of Regulator of G-Protein Signaling-10 in Rats
Authors: Chen Xiaolei , Yu Yang , Bell Balyssa , Cao Yiling , Wang Ye , Felder Robert B , Wei Shun-Guang
Published: September 2019

Systemic pro-inflammatory cytokines (PICs) promote central oxidative stress and neuroinflammation that contribute to sympathetic excitation and cardiovascular dysfunction in hypertension and heart failure. However, the key molecular regulators of neuroinflammatory responses in the brain remain unclear. Regulator of G-protein Signaling-10 (RGS10), a GTPase-accelerating protein that is expressed predominantly in the brain, has recently emerged as an important counter regulator of PICs-induced downstream signaling in the central nervous system. RGS10 negatively regulates NF-kB-mediated inflammatory responses in microglia and antagonizes neurotoxic effects of peripheral inflammation. The present study examined whether peripherally administered PICs alter RGS10 expression in cardiovascular/autonomic brain regions like subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN). Male SD rats received a 2-week subcutaneous infusion of vehicle, interleukin (IL)-1β (400 ng/day) and tumor necrosis factor (TNF)-α (600 ng/day). Rats treated with IL-1β (n=6) or TNF-α (n=6) had significantly (*P<0.05) reduced mRNA level (fold change) of RGS10 in SFO (0.60±0.15* or 0.69±0.16* vs 1.03±0.28; IL-1β or TNF-α, vs vehicle treatment) and PVN (0.72±0.15* or 0.71±0.19* vs 1.02±0.24), and lessoned mRNA level of NF-kB inhibitor IkB-α (SFO: 0.60±0.21* or 0.55±0.16* vs 1.07±0.46; PVN: 0.70±0.31* or 0.82±0.16* vs 1.00±0.07). Treatments with IL-1β or TNF-α increased mRNA level of NADPH oxidase 2 (SFO: 1.96±0.91* or 1.62±0.47* vs 1.03±0.28; PVN: 2.00±0.82* or 1.74±0.70* vs 1.00±0.15) and cyclooxygenase-2 (SFO: 1.78±0.76* or 1.98±1.04* vs 1.00±0.13; PVN: 1.68±0.34* or 1.62±0.57* vs 1.02±0.24). Rats treated with IL-1β or TNF-α had elevated mean blood pressure (98±11* or 100±9* vs 84±5 mmHg) and increased sympathetic activity as indicated by pressure response to ganglionic blockade (38±7 * or 36±6* vs 28±4 mmHg). Additionally, IL-1β or TNF-α-treated rats had increased cardiac fibrotic markers and impaired cardiac diastolic function. These results suggest that circulating PICs reduce RGS10 expression in the brain, which may contribute to sympathetic excitation and cardiovascular dysfunction in inflammatory conditions.
Abstract P2014: Prolyl Endopeptidase (PEP) is the Main Angiotensin (1-7) Forming Enzyme From Angiotensin II (1-8) in the Circulation: Implications for Hypertension
Authors: Gulua Gvantca , Serfozo Peter , Wysocki Jan , Ye Minghao , Liu Pan , Jin Jing , Bader Michael , Myöhänen Timo , Garcia-Horsman J. Arturo , Batlle Daniel
Published: September 2019

The AngII(1-8)-Ang(1-7) axis of the Renin Angiotensin System (RAS) encompasses three enzymes that form Ang(1-7) directly from AngII: Angiotensin Converting Enzyme 2 (ACE2), Prolyl Carboxypeptidase (PRCP) and Prolyl Endopeptidase (PEP). We studied their relative contribution to Ang(1-7) formation in-vivo and also ex-vivo in serum, lungs and kidneys using models of genetic ablation coupled with pharmacological inhibitors. In WT mice infusion of AngII resulted in a rapid increase of plasma Ang(1-7). In a model of ACE2 and PRCP double genetic ablation AngII infusion resulted in a similar increase in Ang(1-7) as in WT (505 ± 46 vs 482 ± 63 pg/ml, respectively), showing that the bulk of Ang(1-7) formation in circulation is essentially independent of ACE2 and PRCP. By contrast, a PEP inhibitor, ZPP, obliterated the rise in plasma Ang(1-7) after infusing AngII(1-8) to control WT mice. In a genetic mouse model of PEP deficiency (PEP
-/-
) the increase in Ang(1-7) was also blunted as compared to WT mice (277±41 and 423±25 pg/ml, respectively p=0.01). The rate of recovery from acute AngII(1-8)-induced hypertension, in PEP
-/-
mice moreover, was delayed as compared to WT mice (Figure). In ex vivo studies PEP inhibition with ZZP reduced Ang(1-7) formation from Ang(1-8) in serum and in lung lysates. By contrast, in kidney lysates the absence of ACE2, but not PEP, obliterated Ang1-7 formation from added AngII. We conclude that PEP is the main enzyme responsible for AngII conversion to Ang(1-7) in the circulation and in the lungs. PEP, not ACE2, is responsible for protecting against AngII-induced hypertension.
Abstract P2017: Angiotensin-Induced Constriction via Rho Kinase Activation in Pressure-Overloaded Rat Thoracic Aortas
Authors: Terada Yuka , Yayama Katsutoshi
Published: September 2019

Activation of the angiotensin II (Ang II) receptor increases myosin light chain (MLC) kinase activity and causes inactivation of MLC phosphatase thorough myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation by Rho kinase. This results in MLC phosphorylation and subsequent smooth muscle constriction. However, the detailed mechanism of Rho kinase activation by Ang II is still unknown. The aim of this study was to clarify whether Ang II-induced constriction in pressure-overloaded rat thoracic aortas is mediated by Rho kinase activation through epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase 1 and 2 (Erk1/2), and janus kinase (JAK).

Pressure-overload in the rat thoracic aortas was produced by suprarenal abdominal aortic coarctation. After 4 weeks, the thoracic aortas were excised for performing organ chamber experiments. Ang II was cumulatively added to reach a final concentration of a 0.1 – 1 μM. The inhibitors were added to 15 min prior to Ang II, and protein levels were measured by Western blotting.

The contractile response to Ang II was significantly enhanced in the pressure-overloaded rats as compared to sham-operated ones [E
max
(% 40 mM KCl-induced constriction): sham; 44.6 ± 2.4, pressure-overload; 61.6 ± 2.2]. Ang II-induced constriction was attenuated by inhibitors of Rho kinase (E
max
: sham; 14.2 ± 5.3, pressure-overload; 6.8 ± 0.6), Erk1/2 (E
max
: sham; 21.2 ± 3.5, pressure-overload; 33.7 ± 3.5), and EGFR (E
max
: sham; 7.8 ± 2.0, pressure-overload; 26.6 ± 3.0) in both the sham-operated and pressure-overloaded rats. The Ang II-induced constriction was attenuated by JAK inhibitor (E
max
: sham; 41.9 ± 4.8, pressure-overload; 45.1 ± 4.2) in only pressure-overloaded rats. The protein levels of MYPT1 and JAK exhibited to increase in the pressure-overloaded rat thoracic aortas (MYPT1: sham; 1.0 ± 0.1, pressure-overload; 1.3 ± 0.1, JAK: sham; 1.0 ± 0.1, pressure-overload; 1.2 ± 0.1).

These data suggest that Ang II-induced constriction is mediated by Rho kinase activation via EGFR, Erk1/2, and JAK in pressure-overloaded rat thoracic aortas. Moreover, Ang II-induced constriction was enhanced in pressure-overloaded rats probably because the protein levels of MYPT1 and JAK tended to increase in the thoracic aortas.
Abstract P2019: (Pro)Renin Receptor-Dependent Induction of Pro-Fibrotic Factors is Mediated by COX-2/EP4/NOX-4/Smad Pathway in Mouse Renal Collecting Duct Cells
Authors: Gonzalez Alexis A , Reyes Cristian
Published: September 2019

The binding of prorenin to the (pro)renin receptor (PRR) stimulates MAPK/ERK1/2, cyclooxygenase-2 (COX-2), NOX-4-dependent production of reactive oxygen species (ROS), transforming growth factor-β (TGF-β), pro-fibrotic factors connecting tissue growth factor (CTGF), and plasminogen activator inhibitor (PAI-I) in collecting duct (CD) cells. However, the specific role of COX-2 and the intracellular signaling pathways involved is not clear. Here, we tested the hypothesis that PRR activation increases profibrotic factors through activation of COX-2-mediated PGE
2
/ E prostanoid receptor 4 (EP4), NOX-4/ROS, and Smad pathway. Activation of PRR in M-1cells treated with recombinant prorenin increased ROS production and protein levels of CTGF, PAI-I and TGF-β. Inhibition of MAPK, NOX-4 and COX-2 prevented these effects. Inhibition of MAKP, COX-2 and EP4 also prevented the upregulation of NOX-4. Because TGF-β activates Smad pathway, we further evaluated phosphorylation of Smad 2/3. Either COX-2 inhibition or EP4 antagonism significantly prevented the phosphorylation of Smad 2/3. Importantly, mice chronically infused with recombinant prorenin (100 ng/ml/h) showed increases in CTGF, PAI-I, TGF- β, fibronectin and collagen I expression levels in isolated CD as well as high expression of alpha smooth muscle actin (α-SMA) and interleukin 1 beta mRNA (IL 1β) in renal tissues, which was ameliorated by concomitant COX-2 inhibition. These results indicate that the induction of TGF-β, CTGF, PAI-I and ROS occurs through PRR-dependent activation of MAPK and NOX-4 via a mechanism that depends on COX-2-derived PGE
2
production, activation of EP4, and Smad pathway.
Abstract P2023: Perivascular Adipose Tissue Assists Arterial Stress Relaxation: Loss of Assistance in Obesity-Associated Hypertension
Authors: Roccabianca Sara , Flood Emma , Garver Hannah , Fink Gregory , Watts Stephanie W
Published: September 2019

In health, PVAT contributes anti-contractile factors, such as adiponectin, that relax the artery. This benefit is lost in cardiovascular disease, including hypertension. We discovered that PVAT’s contributions to arterial function include more than production of vasoactive substances. Using isometric techniques, we tested the hypothesis that PVAT provided structural benefit to the arterial wall by assisting the function of stress relaxation (SR). Arterial rings were mounted with (+) and without (-) PVAT, from the same animal, and taken through a cumulative length tension curve, in which increasing tension (0 - 6 grams) was added. Tissues relaxed for 30 minutes prior to measuring the tension to which the tissue relaxed. Tissues were then challenged with phenylephrine (10
-5
M) for viability at each tension. In thoracic aorta isolated from normal male Sprague-Dawley rats, the presence of PVAT (a brown fat) along the vessel wall assisted SR by reducing the maximum (6 g) cumulative tension achieved (in mgs; -PVAT = 4578±190; +PVAT = 2730±274, p<0.05). Similarly, the PVAT in the superior mesenteric artery (a white fat) promoted SR in a cumulative length tension curve, resulting in a final tension reduced 19% compared to rings -PVAT. The physical association of PVAT with the vessel wall was essential; when PVAT was left only attached to the
ends
of the aortic rings, it provided no benefit in cumulative SR (+/-PVAT = 4122±176). Thus, passive secretion of anticontractile factors does not contribute to stress relaxation. Rather, PVAT must itself be stretched to result in production of humoral factors and/or it alone provides a mechanical benefit. Importantly, PVAT-assisted SR was lost in the thoracic aorta of the hypertensive DahlS rat (systolic BP = 175±9 mm Hg at 17 weeks) fed a high fat diet for 21 weeks (HF -PVAT = 4218±95; +PVAT = 4135±232; p>0.05) when compared to their controls (Con - PVAT = 4422±85; +PVAT = 3706±165; p<0.05; SBP = 111 ± 7 mm Hg). These important findings change how a blood vessel must be considered. PVAT is an essential functional element that should be considered in health and disease.
Abstract P2025: Primary Aldosteronism Decreases Insulin Secretion and Increases Insulin Clearance in Humans
Authors: Adler Gail , Murray Gillian , Turcu Adina , Nian Hui , Yu Chang , Solorzano Carmen , Peng Dungneg , Manning Robert , Luther James M
Published: September 2019

Primary aldosteronism is associated with an increased risk of diabetes, but the mechanism is uncertain. We enrolled 9 patients with primary aldosteronism and assessed insulin secretion and insulin sensitivity beforte and after 3-12 months after either adrenalectomy (n-6) or medical treatment (n=3). Plasma 8AM Aldosterone (
p
=0.02) and 11-deoxycorticosterone (
p
=0.02) decreased after treatment, whereas cortisol was unchanged (
p
=0.57). Systolic blood pressure (131.1±3.6 vs 124.3±7.7;
p
=0.43), serum potassium (3.7±0.1 vs 4.1±0.1 mEq/L;
p
=0.063), and creatinine (1.1±0.1 vs 1.2±0.1 mg/dL;
p
=0.35) were similar after treatment. During hyperglycemic clamps, we infused glucose to increase plasma glucose to a target of 200mg/dL (
a
). The insulin response (
b
, 218.6±39.8 vs 403.1±42.0 pM insulin mean at 90-120 min,
p
=0.004) and L-arginine stimulated insulin response (
c)
increased compared to Pre-Treatment assessment. C-peptide results were similar to insulin alterations. Comparison to control subjects matched for age, race, sex, and BMI (
Figure
, gray line and area indicating mean±95%CI) indicated that insulin secretion after treatment was comparable to healthy individuals. During hyperinsulinemic-euglycemic clamps, insulin clearance (steady state insulin divided by insulin infusion rate) increased after treatment (855±68 vs 615±57 mL/min,
p
=0.031). Insulin sensitivity decreased after treatment (30.7±2.1 vs 18.5±1.6 M/I;
p
=0.016), but disposition index was unchanged (acute insulin response x M/I;
p
=0.81).
Abstract P2036: Effects of Angiotensin II Type 1 A Receptor (AT1aR) on Renal and Urinary Biomarkers of Acute Kidney Injury in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension
Authors: Hosawi Anhar , Dhakal Sanjeev , Thanekar Unmesha , Alawi Laale , Sawant Harshal , Grobe Nadja , Elased Khalid M
Published: September 2019

Renin angiotensin system (RAS) plays a key role in the pathogenesis and progression of hypertension and renal injury. Angiotensin-II (Ang-II) type 1 receptor (AT1R) mediates the actions of Ang II to increase blood pressure, fluid retention, and aldosterone secretion. Ang II is involved in cell injury, vascular remodeling, and inflammation by releasing inflammatory and pro-inflammatory mediators. The effects of Ang II could be counteracted through its catabolism into the vasodilator peptide Ang (1-7) by angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Diabetes and high blood pressure are the major causes of chronic kidney disease which could lead to end stage renal disease. Our previous studies demonstrated that shedding of renal ACE2 and NEP in the urine of
db
/
db
diabetic mice could act as biomarkers for diabetic kidney disease. The aim of the study is to test the hypothesis that there is increased renal ACE2 and NEP shedding in the urine of two-kidney one clip (2K1C) model of renovascular hypertension. In addition, we investigated the effects of deleting AT1aR on these RAS components and on kidney injury molecule-1 (KIM-1). Renovascular hypertension was induced in AT1aR knock out (KO) and WT mice by 2K1C surgery. Blood pressure (BP) measurement by radio-telemetry revealed significant increase of 46.1± 3.6 mmHg in 2K1C mice compared to control mice (p<0.001). Deleting AT1aR significantly decreased the BP in 2K1C mice compared to 2K1C WT mice (69.86 ± 0.19 vs. 151.69 ± 12.22 mmHg, p<0.001). Renal expression and activity of ACE2 and NEP were significantly decreased in the clipped kidney of WT and AT1KO group (p<0.05). In contrast to diabetes, there was no detectable urinary ACE2 and NEP expression and activity in 2KIC mice. In addition, urinary expression of KIM-1 was increased in the clipped kidney. Although deleting AT1R attenuated albuminuria and hypertension, it did not altered urinary expression of KIM-1. These results suggest that the decreased renal NEP and ACE2 in the clipped kidney of 2K1C model may thus worsen kidney injury via impairment of Ang (1-7) formation. In conclusion, urinary KIM-1 could serve as an early indicator of non-diabetic acute kidney injury. Urinary ACE2 and NEP could be used as biomarker of diabetic kidney disease.
Abstract P2037: G Protein-Coupled Receptor 37l1 is Localized on the Nuclear Envelope
Authors: Armando Ines , Jose Pedro A , Konkalmatt Prasad R
Published: September 2019

G protein-coupled receptors (GPCRs), expressed on the plasma membrane, interact with various types of ligands, which trigger a cascade of signal transduction events leading to different intracellular responses that in turn manifest in physiological changes. Some GPCRs also reside and exert signals from intracellular organelles such as nucleus, endoplasmic reticulum, and Golgi apparatus. Recently, we reported that G protein-coupled receptor 37L1 (GPR37L1) is expressed in the apical membrane of renal proximal tubule cells (RPTCs) and participates in luminal sodium transport and blood pressure regulation by regulating the renal expression of NHE3. However, the mechanism by which GPR37L1 regulates NHE3 expression and function in the RPTC has not been studied. Tandem affinity purification of GPR37L1, combined with mass spectrometry analyses, revealed the association of GPR37L1 with mediators of nuclear importing proteins, such as RAN-GTPase, importin-5, and importin-7. In silico analyses of GPR37L1 amino acid sequence revealed the presence of a potential nuclear localization signal at the N-terminus. Confocal fluorescence imaging of RPTCs expressing GPR37L1 tagged with green fluorescence protein (GPR37L1-GFP) showed distinct nuclear membrane expression of GPR37L1. In addition, fluorescence imaging of nuclei isolated from the RPTCs expressing GPR37L1-GFP showed the expression on the nuclear envelope. Immunoblot analyses confirmed the presence of GPR37L1 in the nuclear protein prepared from the RPTCs expressing GPR37L1-GFP. The purity of the nuclear protein preparations was confirmed by the presence of histone deacetylase 2, a marker for nuclear protein, the absence of Na,K-ATPase, a marker for plasma membrane, and the absence of calnexin, a marker for endoplasmic reticulum. Our results show that GPR37L1 also resides on the nuclear envelope and may play a critical role in the regulation of the expression of genes responsible for maintaining normal electrolyte balance and blood pressure.
Abstract P2040: Acute Effects of Weight Loss by Dehydration on Renal Function in Mixed Martial Arts
Authors: Pereira Antonio A , Schor (in memorium) Nestor , Casarini Dulce E
Published: September 2019

Currently there is a significant growth in the world in the number of professional fighters and practitioners of Mixed Martial Arts. In order to promote greater preservation of the physical integrity of the athletes, weight categories were created, however some fighters in the attempt of insertion into inferior categories, use the dehydration for their drastic reduction of the weight. Hypothetically, the method used may trigger some damage to the body, such as a decrease in the volume of renal blood flow. This study aims to identify the acute effects of weight loss by dehydration on kidney function in a Mixed Martial Arts fighter. The study volunteer is MLC, 34 years old, male, black, single, natural and resident in Maceió-AL-Brazil, without metabolic diseases, with a family history of arterial hypertension. It does not use drugs or dietary supplementation. The athlete practices combat sports with participation in competitions in these three years, with an average loss of 7 kg to fight for dehydration during a month before the fights. The signature of the Free and Informed Consent Term of the athletes was collected. Three collections were carried out: one month before the fight, the official weighing day and the day of the event. According to the data presented in the present study, in response to reduction of body weight by dehydration for insertion in a lower fighting category of around 7 kg, it was observed that the athlete presented some abnormalities during the body weight loss / gain cycle as glycosuria, non-reversible positive leukocyturia and proteinuria within 24 hours, elevated serum creatinine (1.3 mg / dL) and urea (42 mg / dL) during the peak of dehydration (day of weighing and fighting). Blood ureic nitrogen / creatinine ratio (15.8) and creatinine clearance (126.1 mL / min) were elevated during the three moments of observation, namely 30 days pre-fight, day of weighing and fighting. Elevated creatinine clearance suggests renal damage with increased filtration rate with possible renal damage from dehydration. We are completing the analysis of a further 10 athletes, and our data suggest the need for guidance of these athletes so that the training method is less detrimental to their physical integrity.
Abstract P2042: Alteration of Mitochondrial Deoxyribonucleic Acid Methylation and Nuclear Transcription Factors Reduces Mitochondrial Biogenesis in Aged Hypertensive Kidney
Authors: Pushpakumar Sathnur , Weber Gregory , Majumder Suravi , Sen Utpal
Published: September 2019

Accumulating evidence suggests that epigenetic mechanisms contribute to aging and target organ injury during hypertension. Aberrant DNA methylation of mitochondrial genes can affect mitochondrial copy numbers, replication and metabolism. Recently, the presence of mitochondrial DNA (mtDNA) methylation was reported; however, its functional significance in aging and hypertension-induced chronic kidney disease is unknown. We hypothesized that alteration in mtDNA methylation aggravates hypertension injury in aged kidney by disrupting mitochondrial homeostasis leading to reduced mitochondrial biogenesis and dysfunction. Hydrogen sulfide (H
2
S) was shown to modulate chromatin recently, we therefore investigated whether H
2
S can act as an epigenetic modifying agent to protect renal mitochondria and improve renal function. Aged (72-75 weeks) and young (10-12 weeks) wild type (WT, C57BL/6J) mice were treated without or with angiotensin-II (Ang II, 1000 ng/Kg/d) and H
2
S donor, GYY4137 (0.5 mg/Kg/d, i.p.), for 28 d. Ang-II increased mean blood pressure in aged mice compared to young (136.54±3.55 vs. 108.68±2.86 mmHg) and reduced glomerular filtration rate (688.3±24.83 vs. 1029.3±30.8 μL/min/100 g b.w.). H
2
S treatment reduced mean BP (122.34±3.47) and increased GFR (962.7±21.5) in aged mice receiving Ang II. In aged mice, querying renal mitochondrial genes for methylation status revealed increased methylation of mt-ND1 and reduction of mt-CO1 and mt-CO2 and H
2
S treatment reversed the changes. Methylation of mt-Cytb was undetected and mt-Dloop was low in Ang II treated aged mice and increased with H
2
S treatment. Ang II treated aged mice exhibited decreased PGC-1α and nuclear transcription factors, NRFI, NRF2, ERRα and TFAM, indicating reduced mitochondrial biogenesis compared to young mice and H
2
S restored their levels. ATP synthase activity, ATP generation was reduced in aged mice receiving Ang II compared to young and increased with H
2
S treatment. Together, our results suggest that alteration in mtDNA methylation and impaired nuclear DNA genes is associated with reduced mitochondrial replication and disrupted metabolism and H
2
S therapy protects the aged kidney from hypertension-induced damage by restoring mitochondrial homeostasis.
Abstract P2043: Role of GRK4, a Risk Factor for Hypertension, in Breast Cancer
Authors: Yue Wei , Wang Jiping , Schiermeyer Katherine , Xu Peng , Gil John , Felder Robin
Published: September 2019

Clinical studies have shown that breast cancer risk is increased in hypertensive women. The molecular mechanism linking hypertension and breast cancer remains undetermined. Several lines of evidence suggest that G-protein coupled receptor kinase 4 (GRK4) is a candidate protein. The link between high salt and hypertension has been well documented. Renal sodium excretion is regulated by dopaminergic D1-like receptor (D
1
R). Sustained activation of GRK4 impairs D
1
R function and causes hypertension. On the other hand, GRK4 expression is higher in breast cancer than in normal breast tissues. GRK4 variants A142V and R65L have been identified as both hypertension and breast cancer risk loci. To determine the role of GRK4 in breast cancer, we first examined GRK4 expression and SNPs in 7 breast cancer cell lines and benign breast epithelial cell line, MCF-10A. We found that MCF-10A cells do not express detectable level of GRK4 protein whereas all 7 breast cancer cell lines express different levels of GRK4. Except for BT20, all other breast cancer lines have 1-3 SNPs on GRK4 gene of which A142V occurs in all 6 lines. MDA-MB-468 cells contain both A142V and R65L at both alleles. These 2 SNPs have been demonstrated to be associated with enhanced GRK4 activity and reduced sodium excretion in the kidney. To further explore the function of GRK4 in breast cancer cells, we generated three stable GRK4 knock-down MDA-MB-468 lines using inducible lentiviral shRNA vectors. GRK4 knock-down induced by doxycycline (Dox) significantly reduced growth rates of these cells with greatest reduction in line 167 (68%). Growth rate changes correlated with that in GRK4 mRNA levels. 5-bromo-2-deoxyuridine (BrdU) incorporation test demonstrated that reduced growth rate was predominantly a result of reduced cell proliferation. Percentage of BrdU labeled cells were decreased from 45±3% in the cells without Dox to 32±5% in the cells treated with 0.1 μg/ml Dox (p<0.005). There was no change in growth rate in the cells expressing non-targeting shRNA. Our results demonstrate that GRK4 plays a role in promoting breast cancer proliferation and suggest that GRK4 could be a new therapeutic target for the treatment of hypertension and breast cancer.
Abstract P2046: The Effect Of Environmental Circadian Disruption On Kidney Injury In Spontaneously Hypertensive Stroke Prone Rats
Authors: Hill Atlantis
Published: September 2019

Obesity, diabetes, and stroke are common morbidities amongst people who engage in shift work. Loosely defined as a lifestyle that includes extended and or rotating shifts, the development of these diseases have been linked to this non-traditional work schedule. Blood pressure like many biological processes are regulated by circadian rhythms. The disruption of these daily rhythms can lead to circadian misalignment where the endogenous rhythms are in discord with the rhythms of the environment. This type of misalignment is a common feature associated with environmental circadian disruption (ECD) and is known to impact several processes in animals and humans. The kidney has been shown both clinically and experimentally to play a unique role in the development of hypertension. Characterized by its ability to maintain body fluid through the balance of water and solutes, the kidney is particularly vulnerable to hypertension. While kidney function is known to exhibit daily rhythms, the relationship between kidney injury and ECD is not well understood. We believe that the disruption of kidney rhythms will lead to an exacerbation of kidney injury in a hypertensive model. In using a spontaneous hypertensive stroke prone rat model, we are able to employ a shift work paradigm to induce ECD. Upon salt loading the animals, we are able to successfully induce hypertension to examine organ damage. To study kidney injury in this model, weekly urine samples were collected to detect the presence of Kidney Injury Molecule-1 (KIM-1) and Albumin. We found that kidney injury was more prominent in the shift group compared to the control group. Leading us to believe that the impact of ECD on the pathology of hypertension may be influenced at the level of the kidneys. These results further confirm accounts in literature that illustrate the role of the kidneys in the development of hypertension and stroke. Our findings suggest that while ECD directly impacts stroke incidence, the kidney may be the target organ in the development of hypertension ultimately increasing the occurrence of stroke.
Abstract P2049: Mitochondrial Respiration And Biogenesis In The Glomeruli Of Dahl Ss Rats
Authors: Sultanova Regina , Domondon Mark , Polina Iuliia , Fomin Mikhail , Stadler Krisztian , Ilatovskaya Daria
Published: September 2019

The mechanisms of salt-sensitive (SS) hypertension are complex and far from clear. Available medications are insufficient to control blood pressure (BP) in the SS subjects, and there is a need for the development of novel effective therapies. SS hypertension is accompanied with an early onset of proteinuria, which results from the loss of podocytes. Many reports highlight that in this disease ultrastructural abnormalities and deficient metabolism in the renal mitochondria may precede histological injury. We hypothesized that podocyte damage occurs in part due to mitochondria dysfunction.

Dahl SS rats were used here as an established animal model of SS hypertension. All rats were fed a 0.4% NaCl (normal salt, NS) diet until 8 weeks of age when they were challenged with a high salt (HS) 4% NaCl diet for 21 days. At the end of the protocol BP was measured, kidneys were cleared from blood, tissues were collected, and cortical glomeruli were isolated and subjected to imaging, WB and other applications.

Systolic BP was elevated in the HS diet fed rats (171±9 vs 148±6 mmHg in HS vs NS diet fed rats). HS diet fed rats exhibited renal lesions, kidney hypertrophy, decreased GFR and exacerbated glomeruli damage compared to the NS group. We did not observe changes in the renal cortical expression of PGC1α; cortical mtDNA content (ND1 and ND6 gene expression vs genomic DNA) was not affected by a HS diet. Seahorse assay performed on freshly isolated glomeruli revealed that basal mitochondria respiration, maximal respiration, and spare respiratory capacity were lower in the HS group (28 ± 14%, 47 ± 15% and 37 ± 9% lower, respectively, vs NS group). No difference was observed in ATP production, or mitochondrial Complex V (ATPase) activity (expressed as oxidation rate of NADH and measured in glomerular pellets). Using confocal imaging and mitoPY1 staining we detected higher baseline H
2
O
2
level, lower antioxidant capacity, and fragmented mitochondria in the podocytes of the glomeruli isolated from HS diet fed group. EM analysis showed that HS mitochondria have structural abnormalities (swelling, enlargement, less defined cristae, and reduced number). We conclude that glomerular mitochondria in SS hypertension are functionally and structurally impaired.
Abstract P2051: Incidence of Renal Artery Damage Following Percutaneous Renal Denervation With Radio Frequency Renal Artery Ablation Systems
Authors: Townsend Raymond R , Walton Antony , Hettrick Douglas A , Hickey Graeme L , Sharp Andrew S
Published: September 2019

Recent prospective randomized clinical trials have demonstrated blood pressure lowering following renal denervation (RDN) with radiofrequency (RF) ablation systems and minimal adverse events in relatively small populations with short follow up duration. The current analysis estimates the rate of renal artery adverse events based on a comprehensive review of published reports. An online database search was performed to identify trials, registries and case reports that employed either the SYMPLICITY Flex or Spyral RF denervation systems (Medtronic, Santa Rosa, CA). Updated reviews of the SYMPLICITY HTN-3 trial and the Global SYMPLICITY Registry were also included. Renal artery stenoses classified as “significant”, greater than 50%, resulted in stent implant or if not specified were counted. The pooled incidence rate of stent implantation was determined by fixed-effects meta-analysis of the study-level incidence rates. Review of 45 published RDN trials reporting on procedural safety with 10,130 patient-years of follow up identified 0.44% of patients (25 of 5,657) with reported renal artery stenosis or dissection and 0.39% of patients (22 of 5,657) with required renal artery stenting. Meta-analysis of all reports indicated an 0.20% pooled annual incidence rate of stent implantation (95% CI: 0.11 to 0.29 % per patient-year). This result was consistent with a fixed effects Poisson regression model (0.22%; 95% CI: 0.14 to 0.32%). An additional 10 case reports including 11 patients with renal artery stenosis resulting in at least one stent implant were identified, with most cases diagnosed after recurrent hypertension, occasionally with concomitant symptoms. Median time from RDN procedure to all renal artery interventions, including both case reports and clinical trials, was 5.5 months (range: 0 to 33 months). A separate review of 14 clinical trials reporting on prospective follow up high resolution imaging of main renal arteries or branches using either MRI, CT or angiography following RDN found a new significant stenosis rate of 0.20% (one event out of 511 patients). Renal artery stenosis and re-intervention following renal denervation with the most commonly applied RF renal denervation system are rare.
Abstract P2052: Differential Association of Measures of Adiposity With 24-Hour Ambulatory Blood Pressure Control in Adults on Antihypertensive Monotherapy
Authors: Cohen Jordana B , Funez Elkys , Cohen Debbie L , Reese Peter P , Townsend Raymond R
Published: September 2019

Background:
Regional measures of adipose distribution, such as visceral adiposity and waist circumference, are more strongly associated with metabolic activity of adipose tissue and longitudinal cardiovascular risk than broader metrics, such as body mass index (BMI) and total fat mass. Little is known about the differential relationship of BMI, waist circumference, visceral adiposity, and total fat mass with degree of blood pressure (BP) control on 24-hour ambulatory BP monitoring (ABPM) in adults with hypertension (HTN).

Methods:
We performed 24-hour ABPM in adults recruited from primary care clinics with a diagnosis of HTN on a stable dose of a single antihypertensive agent. Dual x-ray absorptiometry (DXA) was performed to determine visceral adiposity and total fat mass (indexed by height).

Results:
Of the 97 participants, mean age was 56 (+/- 13) years, with 56 (58%) women, 46 (47%) black, 28 (29%) with diabetes, and 13 (13%) smokers. Mean BMI was 31.7 (+/- 7.2) kg/m
2
and mean waist circumference was 104 (+/- 17) cm. Mean 24-hour systolic BP (SBP) was 135 (+/- 13) mmHg, mean 24-hour SBP average real variability (ARV) was 13 (+/- 3), 47 (48%) were non-dippers, 26 (27%) had masked HTN, and 21 (22%) had uncontrolled HTN. After adjusting for age, sex, race, and antihypertensive class, the highest quartiles of waist circumference and visceral adiposity were associated with higher 24-hour mean SBP (waist circumference β = 9.5, 95% CI 1.9-17.2; visceral adiposity β = 8.6, 95% CI 0.5-16.8) and SBP ARV (waist circumference β = 2.8, 95% CI 1.0 to 4.6; visceral adiposity β = 2.9, 95% CI 1.1 to 4.7) compared to the lowest quartiles; BMI and total fat mass were not associated with these ABPM parameters. Individuals in the highest quartile of visceral adiposity had a 6.3-fold greater odds (95% CI 1.2-33.1) of masked HTN compared to the lowest quartile; no other adiposity measures were associated with masked HTN.

Conclusions:
Elevated visceral adiposity is associated with high 24-hour SBP and SBP variability in individuals with HTN. Unlike other measures of adiposity, higher visceral adiposity is strongly linked with masked hypertension. Measurement of visceral adiposity may help to identify those individuals who would benefit most from 24-hour ABPM to screen for masked hypertension.
Abstract P2060: Life's Simple 7 for Cardiovascular and Total Health: How are We Doing?
Authors: Egan Brent , Li Jiexiang , Sutherland Susan , Jones Daniel , Hong Yuling , Ferdinand Keith , Sinopoli Angelo
Published: September 2019

Background:
Life’s Simple 7 (LS7; diet, physical activity, smoking, BMI, BP, cholesterol, glucose) was developed by AHA to track and improve CV risk and outcomes. Individuals with high vs. low LS7 scores have 30%-80% lower incidence rates of hypertension, diabetes, heart disease, stroke, dementia, kidney and lung disease, and cancer. Since LS7 serves as a population health marker, national trends in demographic groups can inform efforts to optimize health and health equity.

Methods:
LS7 was assessed in 35,562 NHANES 1999-2016 participants 20 years and older, free of CVD with all LS7 data and either non-Hispanic White (NHW), non-Hispanic Black (NHB) or Hispanic. Data were disaggregated by time, age, race / ethnicity and sex. For each LS7 item, 0 points were assigned for poor, 1 point for intermediate, and 2 points for optimal levels; maximum LS7 score = 14.

Results:
LS7 scores fell with age, were higher in NHWs and Hispanics than NHBs, and higher in women than men (Table).

Conclusions:
Demographic factors were associated with large differences in LS7 scores. No group had optimal scores or is improving with time. LS7 scores are sharply lower in middle-age and older than younger adults, in NHBs than NHWs and Hispanics, and in men than women. Hispanics had similar LS7 scores to NHWs, despite less income, education and health insurance. Findings highlight the need for culturally tailored and complementary public and population health programs beginning early in life to promote healthy lifestyle patterns and timely access to and adherence with evidence-based care. Successful programs could reduce the age-related rise in cardiometabolic and other chronic diseases and improve health equity.
Abstract P2062: Vitamin D Supplementation Improves Vascular Function in Adolescents Suffering from Orthostatic Intolerance
Authors: Shaltout Hossam A
Published: September 2019

Background:
In previous work we identified a group of adolescents whose diagnostic workup for chronic unexplained nausea revealed underlying cardiovascular instability manifesting as orthostatic intolerance (OI) and syncope. These patients exhibited impairment in autonomic and vascular function and excessive release of catecholamines upon head up tilt compared to pre tilt. They also had low vitamin D level that correlated with the severity of symptoms on tilt. In this pilot study we hypothesized that vitamin D supplementation to normal levels will improve the vascular function in these participants.

Methods:
A cohort of ten Adolescents (mean age= 16.3 years) who are vitamin D deficient had a head up tilt at baseline and after two months of vitamin D supplementation (2000-5000 IU daily based on baseline level). Heart rate, blood pressure, cardiac work index, arterial compliance and pulse wave velocity (PWV) were measured in supine position.

Results:
As shown in the table. Compared to baseline, vitamin D supplementation improved vitamin D level, arterial compliance and reduced cardiac work index under stress challenge and baseline PWV.

Conclusions:
Vitamin D supplementation for two months restored vitamin D to normal levels and was associated with improvement in the vascular function. These data provides evidence of potential therapeutic benefit of vitamin D supplementation for patients suffering from orthostatic intolerance and syncope who are vitamin D deficient.
Abstract P3002: Endoplasmic Reticulum Stress Plays a Role in Nox5 Mediated Vascular Contraction
Authors: Montezano Augusto C , Camargo Livia L , Kuriakose Jithin , Neves Karla B , Alves-Lopes Rheure , Beattie Wendy , Rios Francisco J , Touyz Rhian M
Published: September 2019

We previously reported that Nox5 regulates contraction through mechanisms involving ROS and Ca
2+
channels in the endoplasmic reticulum (ER). In young mice expressing human Nox5 in VSMCs (Nox5+SM22+), we observed hypercontractility, without changes in blood pressure. Here we tested the hypothesis that Nox5 influences ER Ca
2+
homeostasis and vascular function and that Nox5 amplifies aging-associated vascular dysfunction through processes involving ER stress. Female WT and Nox5+SM22+ mice aged 20 and 35weeks were studied. Blood pressure (BP) was assessed by tail-cuff and vascular function/structure by myography. BP was similar in all groups. Vascular contraction to U46619, a TXA2 analogue, was increased in aged Nox5+SM22+ (EMax - %KCl: 114±2.8 vs WT 95±2.4, p<0.05). Hypercontractility was reversed by NAC (antioxidant - 0.01 mM, EMax: 92±5%), melittin (Nox5 inhibitor - 0.1 μM, EMax 92±3.2%) and dantrolene (RyR Ca
2+
channel blocker - 0.01 mM, EMax: 67±4.2%) (p<0.05). VSMCs isolated from 20 and 35 wk WT and Nox5+SM22+ mice were used to study molecular mechanisms whereby Nox5 influences the contractile machinery, focusing on the ER. Expression of BIP, a marker of ER stress, was increased only in VSMCs from aged Nox5+SM22+ mice (AU: 0.13±0.01 vs WT 0.05±0.002, p<0.05). 4-PBA, an inhibitor of ER stress (1 mM), reversed the hypercontractile responses in 35 wk Nox5+SM22+ mice (EMax: 87±3%, p<0.05). We identified calreticulin, important in ER Ca
2+
homeostasis and channel function, as a molecular target of Nox5. As Nox regulates signalling by oxidation, we assessed calreticulin oxidation by pulldown using dimedone based probe (DCP-Bio). Calreticulin oxidation was increased in mesenteric arteries, aorta and VSMCs from 35 week Nox5+SM22+. Moreover, expression of calreticulin (23.5±2%) and BIP (27.6±9%) was increased by U46619 in VSMCs from Nox5+SM22+ (p<0.05); an effect inhibited by melittin and 4-PBA. Our study highlights molecular mechanisms whereby Nox5 regulates contraction, through oxidation of calcium regulatory proteins, such as calreticulin, and ER stress in aged Nox5 mice. These age related changes may predispose Nox5 mice to cardiovascular damage when challenged with factors associated with hypertension.
Abstract P3003: miR-338-3p Down-Regulation Caused Up-Regulation of Alkaline Ceramidase 2 and Glutathione Peroxidase 3 mRNAs in Subcutaneous Small Arteries of Hypertensive Patients With Chronic Kidney Disease
Authors: Paradis Pierre , Mahjoub Nada , Huo Ku-Geng , Berillo Olga , Richer Chantal , Fraulob-Aquino Júlio C , Rehman Asia , Briet Marie , Boutouyrie Pierre , Lipman Mark L , Sinnett Daniel , Schiffrin Ernesto L
Published: September 2019

Background:
Hypertension (HTN) and chronic kidney disease (CKD) are associated with vascular damage characterized by vascular remodeling and stiffening and endothelial dysfunction. miRNAs are small non-coding RNA that regulate gene expression by binding to their target messenger RNAs (mRNAs), leading to mRNA degradation or translational repression. Their implication in vascular injury remains unclear. We aimed to identify differentially expressed (DE) miRs in small arteries of HTN and CKD human subjects to gain insight into pathophysiological molecular mechanisms in these conditions.

Methods:
Normotensive, HTN [systolic blood pressure (BP) >135 mmHg or diastolic BP of 85-115 mmHg with BpTRU] and CKD subjects (estimated glomerular filtration rate <60mL/min/m
2
) (n=15-16) were studied. Small arteries were isolated from subcutaneous gluteal biopsies and RNA extracted for small and total RNA sequencing using Illumina HiSeq-2500 and further studied using a systems biology approach. Differentially expressed (DE) genes were identified with fold change >1.5 and fold discovery rate <0.1.

Results:
DE miRs were identified (
P
<0.05) uniquely associated with HTN (3↑ and 6↓) or CKD (42↑ and 39↓) or both groups (2↓). Correlation between RNA-sequencing and RT-qPCR data was demonstrated for 3 of 14 tested miRs. Among them, the first-ranked, miR-338-3p (
r
=0.91,
P
<10
-16
), uniquely associated with CKD, was studied further. miR-338-3p and some of its predicted targets were highly expressed in human aortic endothelial cells (HAECs). Two of them, alkaline ceramidase 2 (
ACER2
) and glutathione peroxidase 3 (GPX3), were up-regulated by ~36% and ~60% in HAECs transfected with anti-miR-338-3p, respectively (
P
<0.05). miR-338-3p mimic co-transfection decreased by 30% luciferase activity of reporter vectors containing the conserved wild-type but not mutated
ACER2
or
GPX3
3’ untranslated transcribed region miR-338-3p-5p binding sites (
P
<0.05).

Conclusion:
miR-338-3p was down-regulated in small arteries, which was uniquely associated with CKD.
ACER2
and
GPX3
, identified as miR-338-3p targets, may play a role in vascular injury in CKD.
Abstract P3004: Brain Microvascular Structure During Hypertension: Temporal Changes and Recovery Support Distinct Arteriolar Mechanisms
Authors: Baumbach Gary , Sabharwal Rasna , Gerhold Thomas , Chapleau Mark , Faraci Frank M
Published: September 2019

Hypertension alters vascular structure including diameter, cross-sectional area (CSA), and stiffness of the vessel wall. Inward remodeling is a feature of small vessel disease (SVD), predictive of vascular events and cognitive decline. While effects of prevention of hypertension on large arteries have been studied widely, quantification of temporal changes and recovery from hypertension in microvessels is rare. Previously, angiotensin II (Ang II)-dependent hypertension produced increased CSA but reduced internal diameter (ID) of maximally dilated arterioles (inward remodeling) in brain. Because cerebral arterioles are targets of SVD, we examined time course and whether microvessels recover from hypertension. Mean arterial pressure (MAP) was measured using radiotelemetry in adult male C57BL6J mice prior to, during infusion of vehicle or Ang II (1.4 mg/kg per day using osmotic mini-pumps) for 28 days, and a 28 day recovery period. Prior to treatment, MAP was similar in both groups. Throughout recording, MAP was stable in vehicle treated mice. With Ang II, MAP began to rise on day 3, steadily increasing until day 28. On day 30, MAP began to decrease, reaching levels seen with vehicle on days 46-47. In anesthetized mice, we measured pressure, diameter, and CSA of the vessel wall (histologically) in maximally dilated arterioles (baseline diameter of 63±1 microns) at 1, 3, 7, 14, 28, and 56 days after pump implantation (n=7-9 in each group). At day 1, ID and CSA were similar in both groups. With vehicle, there was no significant change in CSA or ID at any time point. With Ang II, CSA increased at day 7 and was maintained at days 14 and 28 (P=0.023). ID did not change at day 3 or 7, but was reduced (by ~15%, P=0.011) at 14 and 28 days. During recovery (day 56). CSA returned 63% of the way to normal (compared to vehicle), while ID remained at day 14 and 28 values. In conclusion, CSA changed rapidly during hypertension onset and largely recovered with a reduction in MAP. Inward remodeling developed slowly and did not return to vehicle levels with MAP. The lack of recovery of ID after a period of hypertension has implications for the impact of SVD including hypoperfusion, impaired vasodilation, and augmented injury during ischemia.
Abstract P3009: Stenostiffness Approach for Cardiovascular Disease Risk Assessment in Primary Prevention
Authors: Nakano Hiroki , Tomiyama Hirofumi , Yamashina Akira , Study Group J-Babel
Published: September 2019

Background:
The Japan Brachial-Ankle pulse wave VELocity individual participant data meta-analysis of prospective studies (J-BAVEL) studies are a series of individual participant data meta-analyses carried out to demonstrate the predictive ability, individually, of the inter-arm blood pressure difference (IAD), ankle-brachial pressure index (ABI), and brachial-ankle pulse wave velocity (brachia-ankle PWV) for the prediction of cardiovascular events in Japanese subjects.

Purposes:
Based on the findings of the J-BAVEL study series, we discuss here a strategy for integrating IAD, ABI, and brachial-ankle PWV (i.e., steno-stiffness approach) with the conventional cardiovascular disease (CVD) risk prediction model to improve the accuracy of CVD risk prediction in primary prevention.

Methods and Results:
The data of 1,1726 subjects without a past history of cardiovascular disease (CVD) in J-BAVEL study were re-analyzed. The IAD
>
15 mm Hg (IAD15) was associated with a significant Cox stratified adjusted hazard ratio (HR) for subsequent stroke [2.42; 95% confidence interval (CI), 1.27-4.60;
P
<0.01]. The adjusted odds ratio of IAD10 for ABI ≤0.90 in the binary logistic regression analysis was 3.42 (95% CI 2.13-5.50;
P
< 0.001). Furthermore, among 10,512 subjects of J-BAVEL study cohort, subjects having either ABI ≤0.90 {n = 123 (1.2%)} or brachial-ankle PWV ≥15.9 m/sec {n = 3,837 (36.5%)} were defined as abnormal steno-stiffness markers. The addition of abnormal steno-stiffness marker to a model including the FRS improved the accuracy of prediction of the development of CVD (category-free net reclassification improvement: 0.391 [95% CI, 0.266-0.517], P < 0.001; integrated discrimination index: 0.0021 [95% CI, 0.0004-0.0038], P = 0.02).

Conclusion:
Steno-stiffness approach (i.e., IAD, ABI, and brachial-ankle PWV) to CVD risk assessment in primary prevention may be efficient. The IAD is a simply measured useful physiological marker of subclinical PAD (IAD10) or stroke (IAD15). Furthermore, the ABI ≤0.90 or brachial-ankle PWV
>
15.9 m/sec may be efficient for the improvement of CVD risk assessment by conventional risk prediction model in primary prevention.