bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 204: Non-dipper Pattern Is Associated With Urinary Metanephrines In Patients With Refractory Hypertension
Authors: Dudenbostel Tanja , Acelajado Maria C , Li Peng , Oparil Suzanne , Calhoun David A
Published: March 2022

Refractory hypertension (HTN) is an extreme phenotype of treatment-resistant HTN and is defined as blood pressure (BP) ≥140/90 mm Hg on ≥5 antihypertensive drug classes including a diuretic and mineralocorticoid antagonist.

Observational data indicate that African Americans, males, body mass index, kidney function, and diabetes are associated with refractory HTN. Available BP data of refractory HTN are based on clinic or in-home evaluations. Less is known about ambulatory blood pressure (ABP) levels, circadian patterns and pathophysiologic mechanisms.

Aim:
Prospective study of ABP levels, nocturnal BP fall, and urinary (U-) 24-hour metanephrines as index of sympathetic activity.

Methods:
Over a 2 year period, 44 subjects were prospectively enrolled (15 subjects with refractory HTN matched with 29 controlled resistant hypertensive subjects who served as comparator group). All subjects underwent 24-ABP monitoring (ABPM) and U-24-hour metanephrines testing.

Results:
Subjects with refractory HTN were on more antihypertensive drugs (6±1 vs 4.1±1.1, p≤ 0.05) and had significantly higher 24-hour systolic BP(SBP) (174±20.2 vs 139.8±16.3 mm Hg, p=0.0017), higher 24-hour diastolic BP (DBP) (94.7±19.8 vs 75.7±11.8 mm Hg, p=0.006), higher daytime SBP (178.1±97.4 vs 141±15.7 mm Hg, p=0.0046), higher daytime DBP (97.4±19.8 vs 77.2±11.4 mm Hg, p=0.007), higher nighttime SBP (165.2±19.2 vs 133.5±19.8 mm Hg, p=0.0002), and higher nighttime DBP (87.7±16.5 vs 70.2±15.1 mm Hg, p=0.007). Analysis of circadian patterns revealed significant increase in non-dipper pattern, defined as nocturnal BP fall of 0-10%, in subjects with refractory HTN (53.3 % (8 of 15) vs 14.3 % (3 of 28), p=0.0267). Biochemical analysis of U-24-hr fractionated metanephrines revealed significantly higher levels in refractory compared to controlled resistant HTN indicative of higher sympathetic activity (U-24-h total metanephrines (592.2 ±246.9 vs 409.3±167.0 μg, p=0.021, and U-24-h total normetanephrines 464.4 ±250.2 vs 309.8±147.6 μg, p=0.039).

These findings suggest that increased sympathetic activity may play a role in treatment failure and may promote blunted nocturnal BP fall in refractory hypertension.
Abstract 219: Altered Renal and Cardiac Morphology in a Non-Human Primate Model of Spontaneous Essential Hypertension
Authors: Rhoads Megan K , Goleva Slavina B , Beierschmitt Amy , Osborn Jeffrey L
Published: March 2022

Current models of essential hypertension are limited in comparisons to human pathophysiology due to distinct differences in postural positioning, circadian rhythmicity, and genetic variation. We have identified a unique non-human primate model of spontaneous, essential hypertension in
Chlorocebus aethiops sabaeus
, the African Green, or vervet, monkey. Using forearm plethysmography under light ketamine sedation (~15mg/kg), adult vervets were categorized as hypertensive (HT; SBP >140mmHg), borderline hypertensive (BHT; 120mmHg < SBP < 140mmHg), or normotensive (NT; SBP < 120 mmHg). Of the 168 males phenotyped, 32% (53 of 168) were HT (average SBP = 168.79±3.29mmHg), 27% (45 of 168) were BHT (average SBP = 129.58±0.88mmHg), and 41% (70 of 168) were NT (average SBP = 100.21±1.65mmHg). In a smaller study of 18 adult females, 1 was found to be HT (SBP = 156mmHg), 2 were BHT (average SBP = 134±2.0mmHg), and 15 were NT (average SBP = 98.06±4.32mmHg). Fixed paraffin-embedded histological sections of vervet cardiac (H&E) and renal (periodic acid Schiff) tissues were digitized and analyzed. Wall/lumen ratios of renal arterioles (average diameter 33.01±0.5μm) were greater in HT (0.33±0.03) compared to NT (0.24±0.01) male monkeys. Bowman’s capsule space was increased in HT (43.14±3.98% area) glomeruli compared to NT (29.92±2.42% area) glomeruli (p<0.05). Indicative of left ventricular hypertrophy, left ventricular cells were decreased in HT (1833.33±65.48 cells/mm
2
) compared to NT monkeys (2223.61±59.72 cells/mm
2
; p<0.05). The increased wall/lumen ratios and Bowman’s capsular spaces indicate that renal vascular hypertrophy and glomerular damage occur in this non-human primate model of essential hypertension. Left ventricular hypertrophy combined with renal glomerular and vascular dysfunction provides strong evidence for using this non-human primate model as a translational experimental species.
Abstract 226: Adherence to Aspirin Therapy for Primary Prevention of Coronary Artery Disease and Cerebrovascular Accidents
Authors: Imran Tasnim F , Samra Mandeep , Hasan Zachariah I , Thiruvarudsothy Srikanth , Kim Stephanie , Feurdean Mirela , Tentler Aleksey
Published: March 2022

Cardiovascular disease is the leading cause of death in the United States. One simple, overlooked, and easily available therapy can reduce this risk: aspirin. Low dose aspirin reduces the risk of first time myocardial infarction (MI) in men and ischemic stroke (CVA) in women. The American Heart Association recommends anti-platelet therapy for those with an ASCVD (atherosclerotic cardiovascular disease) risk score> 7.5%. The aim of our study was to determine adherence to guidelines regarding aspirin therapy for primary prevention of myocardial infarction and stroke in our resident clinic. A retrospective chart review of randomized patients with risk factors for CAD and CVA seen at Ambulatory Center in Newark, NJ, between 5/1/12 and 10/29/13 was conducted. Those with contraindications to aspirin therapy were excluded.

Over 400 charts were reviewed; 281 met inclusion criteria. Per chart review, only 30% (84/281) of all at-risk patients were taking aspirin. When assessing various risk factors, 44.9% (35/78) of males ages 45-79; 24% (28/116) of females ages 55-79 (95% Confidence Interval [CI] = +-5.9%); 35.7% (51/143) of those with 10-year ASCVD risk score greater than 7.5% (95% CI = +-7.9%); 36.1% (39/108) of diabetic patients (95% CI 95% = +-9.2%); 32.8% (66/201) of those on anti-hypertensive medication (95% CI = +- 6.5%) and 29.3% (36/123) of those with a smoking history were taking aspirin (95% CI = +-8.2%). Of note, the number of women taking aspirin was statistically low as compared to men across all risk factors except for ASCVD score (p<0.01). This study revealed poor adherence to guidelines with regards to use of aspirin for primary prevention of cardiovascular disease and stroke in our resident clinic. In particular, adherence to aspirin therapy for women was strikingly low. However, our estimate may be somewhat low due to improper documentation of over the counter medications. We are implementing interventions to increase awareness of aspirin use such as educational campaigns for patients, mini-lectures for ambulatory resident cohorts, automated EMR reminders and adding aspirin therapy to health maintenance smart texts to achieve our goal of 100% compliance.
Abstract 227: Molecular Mechanism of Arhgef11 via the Rho-Rock Pathway in Promoting Kidney Injury in the Dahl S Rat
Authors: Jia Zhen , Johnson Ashley C , Guo Zibiao , Garrett Michael R
Published: March 2022

We have previously implicated genetic variants in
Arhgef11
, a Rho guanine nucleotide exchange factor, in kidney injury exhibited in the Dahl salt-sensitive (S) rat, a widely studied model of hypertension and chronic kidney disease. We have demonstrated that genetic knockdown (lentiviral shRNA) of
Arhgef11
in HEK293 cells results in reduced RhoA activity (~20%), decreased activation of Rho-ROCK pathway (40-60%), and less stress fiber formation vs. scrambled control. S rat derived primary proximal tubule cells (p-PTC) exhibit increased expression of
Arhgef11
, activation of Rho-ROCK, and when stimulated with the profibrotic cytokine, TGFβ1 are also more prone (vs. control) to epithelial-mesenchymal transition (EMT). These data (along with animal studies) suggest that dysregulation of Rho-ROCK in tubular cells likely play an important role in EMT and promote fibrosis; however, chronic stimulation of this pathway in other kidney cell types including vascular smooth muscle cells (VSMC) may also play a role. Accordingly, S rat primary cultures of VSMC isolated from kidney microvessels (k-VSMC) were studied for differences in the RhoA-ROCK, proliferation, migration, apoptosis, and response to cyclical stretch compared to control k-VSMC (minimal congenic). S rat k-VSMC demonstrated higher expression of
Arhgef11
and activation of Rho-ROCK pathway, similar to p-PTC studies. Cell migration was significantly enhanced (scratch assay) in k-VSMC from S rats (485.0±26.2 nm) compared to 296.2±37.6 nm in cells from control. Apoptosis, evaluated by flow cytometry, was significantly increased in k-VSMC from S rats stimulated with TGFβ1 (10ng/ml, 24hrs), but not Ang II, at both early (9.9%±0.9%) and late apoptosis (22.0%±3.5%) compared to control (7.71%±0.75% and 15.5%±1.3%, respectively). S rat derived k-VSMC also demonstrated up-regulation and activation of Rho-ROCK pathway after cyclical stretch, which models in vivo stimuli such as shear stress. In summary, increased expression of
Arhgef11
(RhoA-ROCK) exhibited by PTC and VSMC cell types in S rat kidney appear to promote epithelial-mesenchymal transition and enhance proliferation, migration, and apoptosis under baseline and profibrotic conditions, which likely contributes to kidney injury.
Abstract 231: Hydrogen Sulfide Promotes Macrophage Polarization to Reduce Renal Inflammation in Angiotensin-II Induced Hypertension
Authors: Pushpakumar Sathnur , Kundu Sourav , Givvimani Srikanth , Sen Utpal
Published: March 2022

Ageing is associated with structural and functional changes in the kidney and the presence of hypertension in this population further contributes to renal complications leading to renovascular sclerosis. Emerging evidence suggests that hydrogen sulfide (H
2
S) pathway participates in the pathogenesis of hypertension. Furthermore, chronic inflammation is known to play a critical role in ageing and development of hypertension. In this study, we hypothesized that angiotensin-II (Ang-II)-induced hypertension causes renal fibrosis by sustained activation of pro-inflammatory M1 macrophage promoting the release of inflammatory cytokines and treatment with H
2
S attenuates renal injury and fibrosis by promoting alternate activation of anti-inflammatory M2 macrophage. C57BL/6J (wild type, WT) mice, aged 18 months, were infused with Ang-II (1000ng/Kg/min) or saline using osmotic pumps for 4 weeks and treated without or with H
2
S in drinking water (30μM/L). Animal groups were 1) Saline, 2) Saline + H
2
S, 3) Ang-II, 4) Ang-II + H
2
S. Blood pressure was measured weekly and renal ultrasound was done to measure hemodynamic parameters. Systolic and diastolic blood pressure was increased in mice receiving Ang-II (160.73 ± 4.5 and 129.10 ± 4.9 respectively) and treatment with H
2
S showed significant reduction (134.5 ± 4.8 and 101.1 ± 4.5 respectively). Ultrasound revealed increased renal artery and intra-renal cortical artery resistive index in Ang-II mice (0.73 ± 0.03) which was significantly decreased following H2S treatment (0.61 ± 0.2). Ang-II increased the protein expression and mRNA levels of pro-inflammatory M1 macrophage marker, CD40, and TNF-α and MCP-1, whereas, H2S treatment increased the expression of anti-inflammatory M2 macrophage marker, CD206, and IL-10. Fluorescent probe H2DCF-DA showed increased intracellular reactive oxygen species with Ang-II treatment which was abrogated with H2S treatment. Our data suggests that H
2
S treatment in Ang-II-induced hypertension attenuates inflammation and hypertension by reducing classical activation of inflammatory M1 macrophage and promotes alternate activation of anti-inflammatory M2 macrophage.
Abstract 251: Dexamethasone Promotes Hypertension In Transgenic Mice By Haplotype-Dependent Regulation Of The Human Angiotensinogen Gene
Authors: Pandey VarunKumar , Kaw Meenakshi , Jain Sudhir , Puri Nitin , Mopidevi Brahmaraju , Rana Anita , Kumar Ashok
Published: March 2022

Human angiotensinogen gene (hAGT) gene has polymorphisms in its 2.5Kb promoter that forms two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C, and -1178G/A). Hap -6A/-217A (Hap -6A) is associated with human hypertension whereas, Hap -6G/-217G (Hap -6G) reduces cardiovascular risk. In vitro, transient transfection shows that Hap -6A has increased promoter activity with a glucocorticoid receptor (GR) agonist (4.7 fold higher) when compared to the Hap -6G. Here, we examine the physiological significance of GR-mediated allele-specific regulation of the hAGT gene, in vivo, with consequential effects on the renin angiotensin system (RAS) in double transgenic (TG) mice engineered with human renin and either haplotype of the hAGT gene. TG mice were treated with and without low dose of a GR agonist, dexamethasone (DEX, 10μg/mL), for 72 hours. In untreated mice, ChIP assay shows greater chromatin-GR binding (Hap -6A- 0.46±0.02 vs. Hap -6G- 0.23±0.01 A.U., p<0.05) and immunoblot shows increased hAGT expression in liver of the Hap -6A-TG mice (Hap -6A- 0.90±0.04 vs. Hap -6G- 0.33±0.03 A.U., p<0.05). However, DEX treatment elicited increased chromatin binding (Hap -6A- 0.80±0.04 vs. Hap -6G- 0.26±0.06 A.U., p<0.05), higher hepatic hAGT expression (Hap -6A- 1.88±0.1 vs. Hap -6G- 0.38±0.09 A.U., p<0.05), increased plasma hAGT (Hap -6A- 1.40±0.11 vs. Hap -6G- 0.42±0.05 A.U., p<0.05) and elevated angiotensin II levels (Hap -6A- 870±45 vs. Hap -6G- 630±47 pg/mL, p<0.05) only in Hap -6A TG mice. No significant change was observed in the endogenous mAGT gene with or without DEX. Similar results were obtained from renal tissues of the two TG lines. Importantly, the GR agonist brought about greater increase in blood pressure (ΔSBP: Hap -6A- 14.5±1.2 vs. Hap -6G- 8.1±1.0 mm Hg, p<0.05) in mice with Hap -6A. Complementary experiments show upregulation of renal redox marker NOX1 (Hap -6A- 1.72±0.13 vs. Hap -6G- 0.45±0.06 A.U., p<0.05) in the -6A TG mice. Taken together, our results show that SNPs in the hAGT Hap -6A favor agonist-induced GR binding that lead to DEX-induced increased expression of the hAGT. This activates the RAS with pathophysiological implications including, increase in blood pressure and tissue oxidative stress.
Abstract 267: Identification And Validation Of New Inhibitors Based On Rational Design Against Dopamine-β-hydroxylase To Combat Hypertension
Authors: Dey Sanjay K , Joseph Toyanji , Kumar Santosh , Kamaladevi A. , Sarkar Nabanita , Thelma B. K , Sarkar Surajit , Balamurugan K. , Kundu Suman
Published: March 2022

Human dopamine β-hydroxylase (hDBH), expressed in noradrenergic nerve terminals of nervous system and in chromaffin cells of adrenal medulla, is a key constituent of catecholamine biosynthetic pathway. DBH inhibition has been shown to help the treatment of hypertension and cardiac heart failure, which are major causes of mortality and morbidity worldwide. Existing hDBH inhibitors are too few, often result in side effects and are frequently non-responsive to specific population. Since no three-dimensional structure existed for full-length hDBH, structure based rational drug design has been elusive till date, an issue to which we provided solution lately by building an experimentally validated in silico model for hDBH. The model was used in Autodock, Glide SP and XP software for structure based virtual screening against small molecule databases from NCI, USA. The docked structures were scored using Autodock, X-Score and Prime MMGBSA. Thus, 69 compounds were identified as prospective inhibitors of DBH, which were then tested in vitro against human serum DBH and its nearly identical homologue, bovine DBH (586 of 617 amino acids homologous), with known inhibitors nepicastat and disulfiram as positive controls. Three lead molecules (NSC637578, NSC99657 and NSC379555) were discovered in the process as potent inhibitors of DBH with IC50s of 1μM, 5.5μM and 18μM, respectively. The binding of the inhibitors to the enzyme were validated using fluorescence and CD spectroscopy as well as ITC, revealing KD values in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated the molecules to be latest generation of DBH inhibitors having very high cell permeability and inability to cross the blood brain barrier. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK 293 cell line and insignificant hemo-toxicities against human RBCs. Hence, in vivo evaluation of the lead molecules has been initiated in model systems like C. elegans and D. melanogaster. These studies reconfirmed their nontoxic properties up to 15μM doses. CombiGlide and Ligand Based Core Hopping methods are being employed to optimize the lead compounds computationally to improve their pharmacokinetic properties.
Abstract 274: Preliminary Candidate Gene Analysis In The Women’S Interagency HIV Study Shows Pleiotropic Effect Of APOB4 And Novel Associations Of BMP4 And NOS3 With Metabolic Syndrome Traits.
Authors: Natanzon Yanina , Zhu Xiaofeng , Morris Nathan , Sharma Anjali , Gustafson Deborah , Liu Chenglong , Greenblatt Ruth , Pearce Leigh , Adeyemi Oluwatoyin , Irvin Ryan , Dykxhoorn Derek , Ofotokun Igho , Floris-Moore Michele , Pierce Chris
Published: March 2022

Purpose:
To evaluate associations between candidate genes that promote pro-vasoconstriction, pro-coagulating, and pro-inflammatory vascular environment which lead to the constellation of phenotypes known as Metabolic Syndrome.

Materials and Methods:
3788 women from the Women’s Interagency HIV Study were genotyped using and Illumina 5M array. Thirty two candidate genes were selected from a comprehensive literature search that promoted pro-vasoconstrictive, pro-coagulative and pro-inflammatory vascular environment. Also three genes (APOB, CETP, LPL) were selected to serve as positive control due to their established association evidence with Metabolic Syndrome and combination of Metabolic Syndrome components. Single SNP regression analysis was performed using the additive model, adjusting for age at baseline and BMI where appropriate. Nine principal components obtained from genome wide SNPs were added to the model to control for population structure. Phenotypes were derived using ATP III definition as a guideline. The baseline values of the phenotype were analyzed. 2000 SNPs were assayed within and 10kb upstream and downstream of each of the 32 gene, which results an experimental wide significance level 2.5e-05.

Results:
Several previously reported SNPs in the APOB gene were marginally significant for HDL cholesterol and total cholesterol traits. Previously unreported SNP rs11687484 reached experimental wide significance for both hemoglobin A1C and fasting glucose. Eight SNPs in CETP reached experimental wide significance with HDL cholesterol, and nonHDL cholesterol/HDL cholesterol ratio phenotypes. A SNP rs72680532 in BMP4 gene is also significantly associated with total cholesterol/HDL cholesterol ratio. BMP4 gene has not been reported to be associated with any Metabolic Syndrome traits or cardiovascular disease. Previously unreported association between SNP rs3918229 in NOS3 gene and HDL cholesterol reached experimental wide significance.

Conclusion:
Multiple genes are associated with traits contributed Metabolic Syndrome, which potentially suggests pleotropic effects.
Abstract 276: Chemerin Decreases Vascular Insulin Signaling.
Authors: Neves Karla B , Lobato Nubia S , Lopes Rheure A , Mestriner Fabiola L , Oliveira Ana Maria , Tostes Rita C
Published: March 2022

Proinflammatory adipokines are key mediators of insulin resistance. The adipokine chemerin influences major aspects of the metabolic syndrome, including adipogenesis and glucose homeostasis in adipocytes and skeletal muscle cells. Amongst its many actions insulin also influences vascular function. Considering that chemerin impairs vascular reactivity, and that its effects in the vascular actions of insulin have not been investigated, we postulate that chemerin decreases vascular responses to insulin by reducing PI3K/Akt signaling. Isometric force was recorded in mesenteric arteries from C57Bl6 mice, incubated with chemerin (0.5 ng/mL, 1 h) or vehicle (veh). Chemerin decreased relaxation responses to insulin (0.1 - 3000 ng/mL, pD2: chemerin: 94.5±0.1 vs. veh: 23.6±0,1) and to ACh (pD2: 5.2±0.1 vs. veh 6.6±0.1). Chemerin effects on insulin-induced vasodilatation were reverted by the PI3K activator YS-49 (pD2: veh= 23.0±0.1; chem= 108.5±0.1, YS-49+chem= 28.5±0.1) and by inhibitors of p38 (pD2: veh= 23.0±0.1; chem= 108.5±0.1, SB203580+chem= 27.1±0.1) and ERK1/2 (pD2: veh= 24.6±0.1; chem= 112.0±0.1, PD98059+chem= 25.4±0.1). Chemerin also decreased PI3K [arbitrary units (a.u.): 0.8±0.1 vs. veh 1.1±0.1) and Akt (a.u.: 0.8±0.1 vs. veh 1.2±0.1) phosphorylation in cultured vascular smooth muscle cells (VSMCs). Furthermore, chemerin inhibited insulin-stimulated glucose uptake by VSMCs ([3H] 2-deoxy-glucose (2DG) uptake, counts per minute: 5668±729.0 vs. veh 9923±662.7). In conclusion, chemerin decreases insulin vascular responses by reducing PI3K/Akt signaling and by activating the MAPK pathway. These results suggest that chemerin may be involved in the pathogenesis of vascular insulin resistance. Our study may contribute to a better understanding of the role of factors released by the adipose tissue on insulin vascular responsiveness and, consequently, on the vascular dysfunction in obesity and obesity-associated diseases. Financial Support: FAPESP.
Abstract 277: Nox Isoforms, Oxidative Stress, Inflammation And Fibrosis In Adipose Tissue In Obese Mice: Differential Regulation In Epididymal, Perirenal And Perivascular Depots.
Authors: Even Sarah E , Montezano Augusto C , Rios Francisco J , Nguyen Dinh Cat Aurélie , Carswell Andrew , He Ying , Antunes Tayze T , Callera Glaucia E , Jandeleit-Dahm Karin , Cooper Mark , Szyndralewiez Cedric , Page Patrick , Touyz Rhian M
Published: March 2022

Adipose tissue inflammation and fibrosis contribute to cardiovascular complications observed in obese patients. Mechanisms related to these deleterious effects are still elusive. Here, we evaluated the oxidative, fibrotic and inflammatory status of obese mice and the putative role of NADPH oxidases. Epididymal (EP), perivascular (PV) and peri-renal (PR) fat were collected from lean (db/m) and obese (db/db) mice. Gene expression was assessed by qPCR, fibrosis by picro Sirius red staining and polarized light microscopy, oxidative stress by Amplex Red (H2O2) and TBARS. In EP fat, white adipose tissue, increased levels of TBARS (56%), CD206 mRNA (macrophage marker - 106%) and fibrosis (300%) were observed in obese mice (p<0.05 vs lean). Similar findings were found in PR adipose tissue, where H2O2 (65%), TBARS (50%), and macrophage infiltration (F4/80 mRNA - 578%) levels were increased (p<0.05 vs lean). In PV fat, mix of white and brown adipose tissue, H2O2 (42%, p<0.05) levels, but not TBARS, were increased. Inflammatory marker mRNA levels such as, CD206 (120%), F4/80 (550%), TNFα (206%), iNOS (46%), as well as collagen 6a (marker of fibrosis), were increased in PV from obese animals (p<0.05 vs lean). Treatment of obese mice with GKT137831 (GKT, Nox1/4 inhibitor, 16 weeks) did not decrease inflammation/fibrosis in EV fat and increased mRNA levels of inflammatory markers in PR adipose depot. GKT decreased mRNA levels of pro-inflammatory markers, followed by an increase in mRNA levels of anti-inflammatory markers, in PV fat. Levels of Nox2 (EP: 1936%, PR: 336%, PV: 358%, p<0.05 vs lean), but not Nox1 or Nox4, were increased in all types of fat; an effect not changed by GKT treatment. In conclusion, oxidative stress is increased in fat from obese animals and may play a role in obesity-associated adipose tissue inflammation and fibrosis. Our data also suggests that Nox1/4 influence adipose inflammation and fibrosis in an adipose tissue-specific manner, with effects being pro-inflammatory in PR fat and anti-inflammatory in PV fat. These phenomena are associated with upregulation of Nox2, possibly due to increased Nox2-containing macrophages. Our findings highlight an important role for Nox isoforms in perivascular adipocyte tissue inflammation/fibrosis in obesity.
Abstract 282: 17β-Estradiol and 16α-Hydroxyestrone Increase Oxidative Stress Through Nrf2 Dysfunction In Human Pulmonary Artery Smooth Muscle Cells - Implications in Pulmonary Hypertension.
Authors: Hood Katie Y , Lopes Rheure A , Johansen Anne K , Montezano Augusto C , Szyndralewiez Cedric , Page Patrick , MacLean Margaret R , Touyz Rhian M
Published: March 2022

Gender differences in pulmonary arterial hypertension (PAH) may be, in part, due to increased formation of the deleterious estrogen metabolite, 16α-hydroxyestrone (16αOHE1). Oxidative stress and Noxs have been implicated in the pathogenesis of PAH. We hypothesised that 17β-estradiol (E2) and 16αOHE1, specifically in human pulmonary artery smooth muscle cells (PASMCs), leads to Nox-induced oxidative stress, which promotes PASMC damage. Cultured human PASMCs were stimulated with either E2 (1nM) or 16αOHE1 (1nM). ROS production was assessed by chemiluminescence (O2-) and Amplex Red (H2O2); antioxidants, regulators of Nrf2, and PCNA (marker of growth) expression by immunoblotting; and Nrf2 activity by ELISA. E2 increased superoxide (219%) and H2O2 (52%) in PASMCs (p<0.05 vs vehicle). E2 induced ROS was blocked by PHTPP (ERβ antagonist), tempol (SOD mimetic), ML171 (Nox1 inhibitor) and GKT137831 (Nox1/4 inhibitor). Thioredoxin (71%), NQ01 (78%) and peroxiredoxin1 (69%) protein levels were decreased by E2, even though Nrf2 activity was increased (38%), p<0.05 vs vehicle. 16αOHE1 exhibited a rapid (5 min) and exaggerated increase in superoxide (355%) and a decrease in H2O2 (65%) production, p<0.05. 16αOHE1-induced ROS was blocked by MPP (ERα antagonist), G15 (GPR30 antagonist), tempol and ML171. 16αOHE1 increased BACH1 (129%; p<0.05), a competitor of Nrf2, which was decreased (92%). E2 stimulation resulted in decreased PCNA expression (30%), while 16αOHE1 increased PCNA levels (150%); p<0.05. E2 and 16αOHE1 induced a rapid and sustained ROS generation in PASMCs derived from PAH subjects. E2 and 16αOHE1 did not increase superoxide production in VSMCs from resistance arteries of healthy subjects. In conclusion, E2 induces ROS production through ERβ-Nox-dependent mechanisms, while 16αOHE1 increases superoxide through an ERα/GPR30-Nox-dependent manner. The effects of E2 and 16αOHE1 on oxidative stress is present in PASMCs but not in VSMCs from peripheral arteries. and seems to be related to a dysregulation of the Nrf2 pathway. These processes may impact on molecular processes contributing to vascular remodeling in PAH.
Abstract 290: Upregulation Of Regulatory T Cells Improves Fetal Development In Preeclamptic Rat Model But Has No Influence On Hypertension And Proteinuria.
Authors: Przybyl Lukasz , Ibrahim Tarek , Haase Nadine , Golic Michaela , Hünig Thomas , Müller Dominik N , LaMarca Babette , Herse Florian , Dechend Ralf
Published: March 2022

Preeclampsia is characterized by hypertension, proteinuria and often associated with intrauterine growth retardation. There is a growing body of evidence that immunological dysregulation, involving T-cells, is implicated in causing preeclampsia. Proper recognition of paternal antigens by regulatory T cells is essential for successful outcome of pregnancy.

The aim of the study was to evaluate the effect of upregulating regulatory T cells in an established transgenic rat model for preeclampsia. Application of CD28-superagonistic antibody has been shown to effectively upregulate regulatory T cells. In the first protocol (treatment arm) we applied 1mg of CD28-superagonist or control antibody on day 11 and 15 of pregnancy. In the second protocol (prevention arm) the superagonist or control antibody was applied on day 1, 5 and 9 to improve placentation with an upregulation of regulatory T cells at implantation site.

Superagonist increased regulatory T cells in circulation and placenta from 1.6 ± 0.2% of CD4-positive T cells to 18.9 ± 4.8% and from 2.61 ± 0.96% to 23.27 ± 7.64%, respectively. Blood pressure (152.1 ± 5.8 mmHg vs 155.1 ± 3.6 mmHg, measured by telemetry on day 16) and albuminuria (30.6 ± 15.1 mg/d vs 14.6 ± 5.5 mg/d) were similar in the superagonist or sham treated preeclamptic group for both protocols. Rats treated with CD28-superagonist, showed higher pup weight (2.66 ± 0.03 g vs 2.37 ± 0.05 g) in preventive protocol and (3.04 ± 0.04 g vs 2.54 ± 0.1 g) in the treatment protocol. Intrauterine growth restriction, calculated by brain to liver weight ratio, was also decreased by superagonist in both protocols (0.88 ± 0.03 vs 1.07 ± 0.03 for prevention strategy and 0.72 ± 0.02 vs 1.05 ± 0.07 for therapeutic protocol respectively). Induction of regulatory T cells in circulation and uteroplacental unit early and late in an established preeclamptic rat model had no influence on hypertension and proteinuria. However, it improved the fetal outcome by ameliorating intrauterine growth retardation substantially.
Abstract 294: CD4
+
T cells Isolated from Preeclamptic Women Produce a Preeclampsia-Like Phenotype in Pregnant Nude-Athymic Rats
Authors: Harmon Ashlyn C , Cornelius Denise C , Wallace Kedra , Moseley Janae , LaMarca Babbette
Published: March 2022

Preeclampsia (PE) is the development of hypertension, with proteinuria, during the second half of pregnancy and is associated with an altered immune function, resulting in a chronic state of inflammation, which is believed to play an important role to increase vasoconstrictors such as Endothelin (ET-1) and autoantibodies as well as much of the pathophysiology associated with PE. It has been previously shown that women with PE have an altered CD4
+
T cell population compared to normal pregnant women. In addition, Ana Zenclussen’s lab has shown that adoptive transfer of activated T helper 1 like splenocytes increased blood pressure, fetal rejection and cytokines secreted from uterine cells when transferred to normal pregnant mice. However, mechanisms causing the increased blood pressure and renal abnormalities were not further investigated. The purpose of this study was to determine if CD4
+
T cells isolated from placentas of PE patients have altered consequences during pregnancy compared to those collected from normal pregnant (NP) women that may further define their role in causing the pathophysiology of PE. CD4
+
T cells were isolated from placentas of PE and NP women, using magnetic anti-CD4 antibodies, cultured in TexMACS medium (Miltenyi Biotec) with IL-2 at 37°C in 5% CO
2
, and injected intraperitoneally into first timed pregnant, nude-athymic rats on day 12 of gestation. On day 18 carotid catheters were implanted and on day 19 MAP was measured and blood and tissues were collected. MAP was significantly increased from 125±3 mmHg in rats injected with NP CD4
+
T cells to 140±9 mmHg in rats injected with PE CD4
+
T cells. Circulating inflammatory cytokines IL-6 and TNF-α were measured and found to be increased in rats exposed to PE vs NP CD4
+
T cells (TNF-α- PE=142.4 pg/mL, NP=79.4 pg/mL; IL-6 - PE=311.6 pg/mL, NP=277.8 pg/mL). Renal cortical ET-1 mRNA expression was found to be increased 4.5 fold in rats exposed to PE CD4+ T cells compared to those exposed to NP CD4+ T cells (p=0.06). These data suggest an important role for placental CD4
+
T cells stimulated in women with PE to cause hypertension and inflammation and renal abnormalities during the course of this disease.
Abstract 295: Proliferation of Endogenous T-reg cells Improves the Pathophysiology Associated with Placental Ischemia of Pregnancy
Authors: Ibrahim Tarek , Przbyl Lukasz , Harmon Ashlyn C , Cornelius Denise C , Moseley Janae , Amaral Lorena M , Faulkner Jessica L , Dechend Ralf , LaMarca Babbette
Published: March 2022

Preeclampsia (PE), new onset of hypertension during pregnancy, is associated with pro-inflammatory cytokines and decreased regulatory immune responses including Tregs and decreased IL-10. We believe this decrease in immune regulatory mechanisms leads to much of the pathophysiology associated with PE such as elevated blood pressure and decrease in fetal weight. The RUPP rat model of induced placental ischemia exhibits similar characteristics as women with PE regarding high blood pressure, cytokine levels and immune cell activation and decreased Tregs and pup weight. Therefore, we hypothesized that administration of a CD28 superantagonist (SA) would increase the Treg profile in the RUPP rats which could reduce pro-inflammatory cytokines and blood pressure. Chronic Reduced Uterine Perfusion Pressure, the RUPP procedure, was performed at gestation day 14 (GD14); SA was administered intraperitoneally at GD15, GD18 carotid catheters inserted, and GD19 MAP and pup weight, serum and tissues were collected. MAP in NP rats was 99.5 +/- 2.1, in 116.6 +/- 2.04 in RUPPs which decreased to 107.8 +/- 2.1 mmHg in RUPP+SA. Circulating FoxP3+ Treg cells were undetectable in RUPP rats but increased to 10.96% in RUPP+SA. IL-10 was 24.6 +/- 13 in NP, 35.6 +/- 17 in RUPP and 158.7 +/- 120 pg/mL in RUPP+SA; IL-2 was 6.8 +/- 6 in NP, 89.4 +/- 24 in RUPP, and 126.3+/- 23.7 pg/mL in RUPP+SA. Pup weight was 2.135 +/- 0.23 in NP, 1.964 +/- 0.13 in RUPP, but increased to 2.127 +/- 0.13 mg in RUPP+SA. These data suggest an important role for up-regulating Treg cells to enhance the immune regulatory interactions and inhibit the hypertension while safely improving pup weight in response to placental ischemia during pregnancy.
Abstract 297: Naturally Occurring Cullin-3 Mutations Decrease Substrate Ubiquitination and Acts Dominantly by Sequestering Substrate Adaptors for Cul3
Authors: Ibeawuchi Stella-Rita C , Agbor Larry N , Quelle Frederick W , Sigmund Curt D
Published: March 2022

Cullin-Ring ubiquitin Ligases (CRL) regulate protein turnover by promoting ubiquitination of substrate proteins targeting them for proteosomal degradation. Impaired Cullin-3 (Cul3) activity in vascular smooth muscle causes increased RhoA protein and Rho kinase signaling leading to impaired vasodilation and augmented contraction. PHAII patients with de novo mutations in Cul3 which cause skipping of exon 9 in Cul3 (Cul3Δ9) have early onset hypertension, but the mechanistic basis remains unclear. We hypothesize that Cul3Δ9 results in reduced RhoA ubiquitination and degradation, increasing RhoA activity. We cloned a mutant Cul3Δ9 cDNA by “splicing by overhang extension” PCR. Endogenous RhoA protein was increased in HEK293T cells expressing Flag-Cul3Δ9 compared with cells expressing wildtype Flag-Cul3 (Cul3WT). There was no defect in neddylation of Cul3Δ9. However, binding of Cul3Δ9 to RBX1, which is necessary for delivering ubiquitin to the substrate was impaired. Consistent with this, ubiquitination of epitope-tagged RhoA by Cul3Δ9 was significantly impaired compared with Cul3WT both in vivo and in vitro. Thus Cul3Δ9 exhibits impaired ubiquitination of Cul3 substrates. Interestingly, co-IP studies revealed that the substrate adaptor proteins Bacurd1, RhoBTB1 and KLHL3 bound more efficiently to Cul3Δ9 than Cul3WT. Moreover, we observed a significant increase in Cul3Δ9:Cul3WT heterodimers compared to Cul3Δ9:Cul3Δ9 homodimers. This is important because dimerization of Cul3 ubiquitin ligase complexes is required for efficient ubiquitination of some substrates. Thus Cul3Δ9 may act dominantly by interfering with Cul3WT by sequestering substrate adaptors from the CRL3 complex or by directly inhibiting the Cul3 dimer. We conclude that Cul3Δ9 exhibits impaired ubiquitin ligase activity and interferes with activity of Cul3WT. In smooth muscle, this would increase the RhoA pool that can be activated in response to contractile agonists. When combined with our previous data, we suggest that Cul3-mediated regulation of RhoA protein turnover controls both vascular function and arterial blood pressure. Patients carrying Cul3Δ9 mutations may exhibit elevated smooth muscle RhoA/Rho kinase activity contributing to their hypertension.
Abstract 300: The Notch Signaling Pathway Regulates the Fate of Renal FOXD1+ Stromal Cells and Their Descendants
Authors: Lin Eugene E , Pentz Ellen S , Li Minghong , Sequeira-Lopez Maria Luisa S , Gomez R. A
Published: March 2022

The mechanisms underlying the establishment, assembly and maintenance of the renal blood vessels are poorly understood. We have previously shown that renal stromal cells, characterized by their early and transient expression of the transcription factor Forkhead box D1 (FOXD1), are precursors for several cell populations of the renal vasculature, including arterial smooth muscle cells (SMCs), renin cells, pericytes, and mesangial cells. To better understand the role and fate of FOXD1
+
stromal cells, we isolated FOXD1
+
cells and their descendents. Mice with cre recombinase knocked into the FOXD1 locus (FOXD1-cre) were crossed with mice carrying the mT/mG reporter, which exchanges RFP expression for GFP upon cre-mediated recombination. GFP-positive FOXD1-lineage cells were isolated from the kidney using FACS. These cells comprised on average 19% (n=8) of adult kidney cells. As expected, these cells expressed markers of SMCs (α-smooth muscle actin) and renin cells (renin), as shown by RT-PCR. Interestingly, the cells did not express markers of endothelial cells, including Tie2 and β-globin. Prior data from our lab also demonstrated that the transcription factor “Recombination signal binding protein for immunoglobulin kappa J region” (RBP-J), the final transcriptional mediator of Notch signaling, is expressed in FOXD1-lineage cells and plays a key role in their differentiation, as deletion of RBP-J using FOXD1-cre led to severe reductions of the endowment of FOXD1
+
descendants, with subsequent renal abnormalities including glomerular aneurysms, and vessel fibrosis. To better understand this phenotype, we wished to identify the Notch-pathway receptors and ligands expressed in FOXD1-lineage cells. RT-PCR analysis showed the expression of Notch 3 and Jagged 2 in both adult and newborn (postnatal day 1) animals, and Notch 2 and Jagged 1 exclusively at the newborn stage. Deletion of Notch 1 or 2 within FOXD1 cells failed to reproduce the phenotype of RBP-J deletion. However, mutant animals exhibited glomerulosclerosis and regions of fibrosis. In addition, a small number of arteries appear to enter the renal parenchyma from the subcapsular region, an inversion of the normal pattern, indicating a possible role for the Notch receptor in renal vessel guidance.
Abstract 322: Protective effects of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on renal damage in Dahl Salt-Sensitive Rats
Authors: Worou Morel E , D’Ambrosio Martin , Liao Tang-Dong , Nakagawa Pablo , Rhaleb Nour-Eddine , Carretero Oscar A
Published: March 2022

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide hydrolyzed by angiotensin-converting enzyme. We previously reported that Ac-SDKP protects the kidney from inflammation, fibrosis, and dysfunction caused by hypertension. Salt-sensitivity is highly prevalent in African-Americans and elderly patients, and is associated with a high rate of hypertensive renal disease.

The effect of Ac-SDKP on Salt-Sensitive (SS) hypertension is unknown; here, we hypothesized that Ac-SDKP prevents renal fibrosis, glomerulosclerosis and albuminuria in Dahl SS rats on high salt diet without affecting blood pressure.

Dahl SS rats and their consomic control, SS-13BN were fed either 0.23% NaCl (low salt, LS) or 4% NaCl (high salt, HS) diet and infused with Ac-SDKP (800 μg/kg/day, subcutaneously
via
osmotic minipump) or vehicle (0.01 N acetic acid) for 6 weeks. Animals were divided into following groups: LS+vehicle, HS+vehicle, and HS+Ac-SDKP.

High salt increased systolic blood pressure (SBP) in SS rats (HS+vehicle 186±5 vs. LS+vehicle 141±3 mmHg), but not in SS-13BN (HS+vehicle 140±3 vs. LS+vehicle 140±2 mmHg). Ac-SDKP did not affect SBP. In both SS and SS-13BN, HS induced renal and cardiac hypertrophy, which were not prevented by Ac-SDKP. However, in SS rats, Ac-SDKP prevented HS-induced renal interstitial fibrosis (HS+vehicle 12±0.5 vs. HS+Ac-SDKP 7±1 % of renal interstitial area) and glomeruloslerosis (HS+vehicle 18±1 vs. HS+Ac-SDKP 9±1 % of glomerular tuft area). Compared to LS, HS increased albuminuria in both strains, from 13±1 to 94±10 mg/24h in SS and from 7±1 to 44±8 mg/24h in SS-13BN. HS also decreased nephrin protein expression in both strains. Interestingly, in SS-13BN, Ac-SDKP attenuated albuminuria (24±3.18 mg/24h) and partially attenuated the decrease in nephrin expression, but in SS, Ac-SDKP had no effect on either albuminuria (126±34 mg/24h) or nephrin expression.

In summary, Ac-SDKP prevents renal fibrosis, glomerulosclerosis but not albuminuria in Dahl SS rats on high salt diet. On the other hand, the consomic controls fed high salt developed albuminuria and decreased nephrin, and these effects were prevented by Ac-SDKP.
Abstract 326: Salt Intake, Drinking Behavior, And The Body Corporate
Authors: Rakova Natalia , Lerchl Kathrin , Dahlmann Anke , Rauh Manfred , Goller Ulrike , Basner Mathias , Dinges David F , Beck Luis , Agureev Alexander , Larina Irina , Baranov Victor , Morukov Boris , Eckardt Kai-Uwe , Vassilieva Galina , Wabel Peter , Vienken Jörg , Kirsch Karl , Johannes Bernd , Luft Friedrich C , Titze Jens
Published: March 2022

Clinical practice and public health advice on salt consumption relies on the generally accepted notion that accurately collected 24-hour urine collections are valid estimates for salt and fluid intake. Reduced salt intake would also reduce thirst and ingestion of sugary drinks accordingly. To test these widely accepted ideas, we performed two independent ultra-long term salt balance studies lasting 105 and 205 days in 10 men simulating a flight to Mars. We fixed dietary intake of all constituents for months at salt intakes of 6, 9, and 12 grams per day and collected all urine. We controlled all environmental factors for rigorous quantitative comparison of urine volume and salt excretion with daily fluid and salt intake. In 1646 collected 24-hour urine samples, our ten subjects recovered 92% of salt they ingested. Due to infradian rhythmical renal salt excretion, only 781 out of 1646 daily urine samples correctly classified a 3 g difference in salt intake (47%). Reducing salt intake from 12 to 6 g reduced 24-hour cortisone excretion (78.3±19.6 vs. 67.2±19.3 μg/d; P<0.001) and increased water intake by 299 ml/d due to reduced glucocorticoid-driven metabolic water production. Even accurately collected 24-hour urine collections at intakes ranging from 6-12 grams salt per day provide no reliable information on individual salt intake. Reducing salt intake leads to reduced glucocorticoid levels decreased metabolic water production and a counterintuitive increase in fluid intake. Traditional views on fluid and electrolyte homeostasis may lead to misinterpretations in clinical practice, public health research, and policy-decision making.
Abstract 327: The Choice of Diuretic is Important in the Treatment of Resistant Hypertension
Authors: Judd Eric , Colon Chad M , Cutter Gary , Calhoun David A
Published: March 2022

Background:
Diuretic therapy is a critical part of the antihypertensive regimen in patients with resistant HTN; however, the ideal diuretic regimen has not been defined. We hypothesized that BP response is dependent on diuretic type. In the current study, BP response with hydrochlorothiazide (HCTZ), chlorthalidone, and loop diuretics was compared for treatment of resistant HTN.

Methods:
Data from clinic visits of adults who were referred for resistant HTN to the HTN Clinic at the University of Alabama at Birmingham between January 2008 and May 2013 were analyzed. Changes in office BP by diuretic type were assessed using a mixed linear model to account for multiple clinic visits with covariate adjustment. Aldosterone status and dietary sodium/potassium were tested for associations with response to diuretic therapy in a subset of patients with complete 24 hour urine collections (n = 131).

Results:
The study population consisted of 342 patients with 963 clinic visits; 153 out of 342 patients were Black and 202 were women. The median BP on presentation was 152/83 mm Hg while taking 4 (median) classes of antihypertensive medications. Over the course of a median of 3 visits the presence of a diuretic was associated with a lowering in office systolic BP (p = 0.001) after adjusting for age, race, number of medication classes, time between visits, spironolactone use, and CKD stage. However, analyzed by diuretic type, chlorthalidone significantly reduced office BP (p < 0.0001) while HCTZ and loop diuretics did not (p = 0.6 and p = 0.9, respectively) after adjustment for covariates. In the adjusted model, an increase in chlorthalidone dose by 12.5mg was associated with a 5.0 mm Hg reduction in office systolic BP. The BP response to chlorthalidone did not vary by race (p = 0.3), dietary sodium-to-potassium intake (p= 0.3), or aldosterone status (p = 0.4), yet was reduced by increasing CKD stage (p = 0.01).

Conclusions:
Chlorthalidone was shown to affect BP response in resistant HTN with significant reductions in BP, while HCTZ and loop diuretics were not. The response to chlorthalidone was not explained by race, dietary sodium-to-potassium ratio, or aldosterone status.
Abstract 329: Blood Pressure Variability during High Salt Intake Is Associated with Cardiovascular Events in Patients with Essential Hypertension
Authors: Kisaka Tomohiko , Ozono Ryoji , Cox Timothy A , Sasaki Nobuo , Ishida Takafumi , Higashi Yukihito , Oshima Tetsuya , Kihara Yasuki
Published: March 2022

Introduction:
Accumulating evidence suggests that blood pressure variability (BPV) is a strong predictor of cardiovascular events in patients with hypertension. However, it is unknown whether high salt intake may affect BPV and its predictive value.

Method:
We analyzed the prevalence of cardiovascular events in 47 patients with essential hypertension in whom systolic BPV was determined during three different 7 day salt intake diets: regular (10g/day NaCl diet), low (3g), and high (20g). Supine blood pressures were measured every hour from 6:00 through 23:00. Mean and standard deviation (SD) in the supine systolic blood pressure (SBP) was used to calculate BPV.

Results:
SDs in SBP during the regular salt (12.5 ± 4.8 mm Hg) and high salt (12.8 ± 5.4 mm Hg) diets were significantly increased compared with the low salt (10.2 ± 4.0 mm Hg). Coefficients of variation (CV) in SBP for the regular, low, and high salt diets were 8 ± 3%, 7 ± 2% and 8 ± 3%, respectively. Salt loading significantly increased both SD and CV (p < 0.05). During a 22 ± 6 year follow-up period, 25 cardiovascular events were observed including 9 fatal events. ROC analyses revealed SD of SBP during regular salt diet had an AUC = 0.66 (95% CI, 0.53 - 0.74) and high salt diet of AUC = 0.72 (0.58 - 0.79), but the low salt diet was not associated with adverse events. CV of SBP during high salt diet, AUC = 0.72 (0.60 - 0.79), was also significantly associated with events. Kaplan Meier analysis also showed that patients with CV of SBP larger than 8% had significantly greater number of cardiovascular events (p < 0.05).

Conclusion:
These results suggest that high salt diet increased BPV, thereby leading to increased incidence of cardiovascular diseases.