bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in Cytokines, Haemodynamics and Microcirculation in Patients with Sepsis/Septic Shock Undergoing Continuous Renal Replacement Therapy and Blood Purification with CytoSorb
Authors: Zuccari Samuele , Damiani Elisa , Domizi Roberta , Scorcella Claudia , D’Arezzo Mario , Carsetti Andrea , Pantanetti Simona , Vannicola Sara , Casarotta Erika , Ranghino Andrea , Donati Abele , Adrario Erica
Published: September 2019

Background: Extracorporeal blood purification therapies have been proposed as a strategy to remove inflammatory mediators during sepsis, thus improving outcome. Objectives: We aimed to evaluate changes in cytokines, haemodynamics and microcirculation during blood purification with Cytosorb adsorber in septic patients. Methods: Prospective observational study on critically ill adult patients with sepsis/septic shock underwent renal replacement therapy (RRT) for acute renal failure and haemoadsorption with Cytosorb as adjunctive therapy for 24 h. Measurements were taken at baseline, after 6 and 24 h: haemodynamic parameters, arterial and central venous blood gases, plasma levels of tumour necrosis factor alpha, interleukin (IL) 1-beta, IL-6, IL-8 and IL-10. The sublingual microcirculation was assessed with sidestream dark field videomicroscopy to evaluate the perfused vessel density (PVD) and microvascular flow quality. Tissue oxygenation and microvascular reactivity were assessed with thenar near infrared spectroscopy (NIRS) with a vascular occlusion test. Results: Nine patients; plasma levels of IL-8 decreased at 24 h (p < 0.05 versus 6 h); no significant variation was found for other cytokines. Haemodynamic remained stable throughout the observation. Microvascular perfusion improved over time, with an increase in PVDs at 6 and 24 h (from 13.9 [13.3–16.4] to 15.7 [15–17.3] and 17 [14.8–18.6] mm/mm2 respectively, p = 0.003) and total vessel densities at 24 h (14.9 [13.9–16.9] vs. 17.9 [15.3–20], p = 0.0015). No significant variation was detected in NIRS-derived parameters. The Sequential Organ Failure Assessment score decreased from 12 ± 3 to 10 ± 1 at 24 h (p = 0.039). Conclusions: In septic patients undergoing RRT, haemoadsorption with Cytosorb seems to determine a decreasing in plasma levels of IL-8, although levels of other cytokines did not vary significantly, and an improvement of microcirculation despite no significant variation in macro-haemodynamics.
Benefits and Complications during the Stay at an Early Rehabilitation Facility after Radical Cystectomy and Orthotopic Ileum Neobladder Reconstruction
Authors: Schulz Gerald B. , Grimm Tobias , Buchner Alexander , Kretschmer Alexander , Stief Christian G. , Karl Alexander , Jokisch Friedrich
Published: September 2019

Purpose: Early rehabilitation (ER) after radical cystectomy (RC) seems to be crucial for quality of life, education and prevention of complications after hospital discharge. We investigated an inpatient ER setting for bladder cancer (BC) patients. Methods: In total, 103 BC patients who underwent ileum neobladder reconstruction were included. The major issues from the patients’ point of view, functional outcome parameters and complications during ER were analysed. A Wilcoxon signed rank test was used to compare body mass index (BMI) and diurnal as well as nocturnal use of urinary pads before and after ER. Results: At the beginning of ER, the median Karnovsky performance scale score was 70% (interquartile range [IQR] 70–90%) and the mean BMI was 25.8 kg/m2 (IQR 21.9–27.9). The 4 most common complaints were urinary incontinence (80.6%), general weakness (73.8%), urinary mucus (49.5%) and mental distress (44.7%). During the programme, 28.2% of patients had a urinary tract infection requiring antibiotics and 15.5% presented a symptomatic acidosis. Median diurnal use of urinary pads significantly decreased during ER (4 vs. 3; p < 0.001). At the end of the ER programme, 76.0, 54.8 and 30.8% of the patients indicated an improvement of their physical capacity, incontinence and psychological distress respectively. Conclusions: Our study demonstrates the need for postoperative rehabilitation after RC. Further investigations should compare outcome parameters to ambulatory and outpatient ER models.
Epiploic Appendagitis: An Often Misdiagnosed Cause of Acute Abdomen
Authors: Suresh Kumar Vishnu Charan , Mani Kishore Kumar , Alwakkaa Hisham , Shina James
Published: September 2019

Epiploic appendages are peritoneal structures that arise from the outer serosal surface of the bowel wall towards the peritoneal pouch. They are filled with adipose tissue and contain a vascular stalk. Epiploic appendagitis is a rare cause of acute lower abdominal pain. It most commonly results from torsion and inflammation of the epiploic appendages, and its clinical features mimic acute diverticulitis or acute appendicitis resulting in being often misdiagnosed as diverticulitis or appendicitis. This frequently leads to unnecessary hospitalization, antibiotic administration, and unwarranted surgeries. Epiploic appendagitis is usually diagnosed with CT imaging, and the classic CT findings include: (i) fat-density ovoid lesion (hyperattenuating ring sign), (ii) mild bowel wall thickening, and (iii) a central high-attenuation focus within the fatty lesion (central dot sign). It is treated conservatively, and symptoms typically resolve in a few days. Therefore, epiploic appendagitis should be considered as one of the differential diagnosis for acute lower abdominal pain and prompt diagnosis of epiploic appendagitis can avoid unnecessary hospitalization and surgical intervention. In this case report, we discuss a 72-year-old woman who presented with a 2-day history of acute left lower abdominal pain.
Early Copeptin Determination Allows Prompt Diagnosis of Post-Neurosurgical Central Diabetes Insipidus
Authors: Berton Alessandro Maria , Gatti Filippo , Penner Federica , Varaldo Emanuele , Prencipe Nunzia , Rumbolo Francesca , Settanni Fabio , Gasco Valentina , Ghigo Ezio , Zenga Francesco , Grottoli Silvia
Published: September 2019

Introduction: Central diabetes insipidus (CDI) is a frequent complication of pituitary surgery, but its diagnosis lacks standardized criteria. Copeptin, a surrogate marker of arginine vasopressin release, is triggered by psycho-physical stresses such as pituitary surgery. Low postoperative copeptin could predict CDI onset. The aims of this study were the validation of copeptin as a predictor of post-neurosurgical CDI and the identification of the optimal timing for its determination. Methods: Sixty-six consecutive patients operated for a hypothalamic-pituitary lesion were evaluated. Copeptin was determined preoperatively and at 1, 6, 12, 24 and 48 h post-extubation. Fifty-eight patients were reassessed after 3–6 months post-surgery to confirm transient (3 cases) or permanent CDI (5 cases) diagnosis. Results: A marked copeptin peak was identified at 1 h after extubation, when a value below or equal to 12.8 pmol/L had a good accuracy in identifying CDI cases (AUC 0.866, 95% CI 0.751–0.941). Moreover, a copeptin peak above 4.2 pmol/L excluded permanent forms (AUC 1, 95% CI 0.629–1). Regression analysis identified copeptin as the only significant predictor of CDI (OR 0.86, 95% CI 0.75–0.98, p = 0.02). A copeptin T1/T0 ratio below or equal to 1.47 identified patients at risk of isolated biochemical alterations even in the absence of an overt CDI. Conclusions: A prompt increase of copeptin is expected at 1 h after extubation. The absence of this peak is a reliable predictor of post-neurosurgical CDI.
Hypothalamic Structural and Functional Imbalances in Anorexia Nervosa
Authors: Florent Vincent , Baroncini Marc , Jissendi-Tchofo Patrice , Lopes Renaud , Vanhoutte Matthieu , Rasika Sowmyalakshmi , Pruvo Jean-Pierre , Vignau Jean , Verdun Stephane , Johansen Jeanette E. , Pigeyre Marie , Bouret Sebastien G. , Nilsson Ida A.K. , Prevot Vincent
Published: September 2019

The hypothalamus contains integrative systems that support life, including physiological processes such as food intake, energy expenditure, and reproduction. Here, we show that anorexia nervosa (AN) patients, contrary to normal weight and constitutionally lean individuals, respond with a paradoxical reduction in hypothalamic levels of glutamate/glutamine (Glx) upon feeding. This reversal of the Glx response is associated with decreased wiring in the arcuate nucleus and increased connectivity in the lateral hypothalamic area, which are involved in the regulation on a variety of physiological and behavioral functions including the control of food intake and energy balance. The identification of distinct hypothalamic neurochemical dysfunctions and associated structural variations in AN paves the way for the development of new diagnostic and treatment strategies in conditions associated with abnormal body mass index and a maladaptive response to negative energy balance.
Abstract 013: Racial Disparities in Maternal Hypertension and Pregnancy Outcomes a Population-Based Study
Authors: Bhatt Chintan , Saxena Anshul , Rubens Muni , Armaignac Donna , Salami Joseph , Das Sankalp , Veledar Emir
Published: September 2019

Objective:
Maternal pre-pregnancy hypertension is an established risk factor for preterm births and infant deaths. We sought to investigate the effects of maternal pre-pregnancy hypertension and tobacco use on preterm and infant deaths.

Methods:
We analyzed National Vital Statistics System’s 2013 and 2015 linked birth/ infant death data from a total sample of 6,629,652 live births. There were 11,782 preterm deaths due to “Newborn affected by maternal factors and by complications.” Maternal factors and complications were defined as deaths at less than 1 year of age with
ICD-10 codes
P00-P04. Regression model was accounted for tobacco use, cigarette use before and during pregnancy, prenatal care, maternal age and demographics.

Results:
Disease specific preterm mortality due to pre-pregnancy hypertension was 1.9 per/1000 live births. Preterm risk increased by two folds (adjusted odds ratio (AOR)=2.12; 95% confidence interval (CI) 1.92-2.18) with maternal pre-pregnancy hypertension history. Presence of pre-pregnancy hypertension and tobacco use doubled the risk of preterm (AOR=3.82 (95% CI: 3.67-4.82). African American mothers with pre-pregnancy hypertension were 1.72 time more likely to have preterm mortality than White females.

Conclusion:
Black race, maternal risk factor pre-pregnancy hypertension and tobacco use during pregnancy were associated with higher risk of preterm mortality. Further investigation is crucial to better understand the risk factors for the disparities in preterm so that women who are at risk pre-pregnancy can be identified and provided risk-specific services.
Abstract 022: Role of Interleukin-17 in the Regulation of Hemodynamics and Renal Sympathetic Nerve Activity in Rats
Authors: Cao Yiling , Yu Yang , Wang Ye , Felder Robert B , Wei Shun-Guang
Published: September 2019

Introduction:
Central inflammation has been implicated in the pathophysiology of hypertension (HTN) and heart failure (HF). We previously reported that the pro-inflammatory cytokines (PIC) tumor necrosis factor-α and interleukin (IL)-1β act within the brain to increase blood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) and contribute significantly to the neurohumoral activation in these diseases. Over the years, IL-17A, a PIC secreted mainly by Th17 cells and γδ T cells, has been identified as a critical inflammatory mediator promoting chronic inflammation in cardiovascular diseases. Growing evidence indicates that IL-17A penetrates the blood-brain barrier and that its receptors, IL-17RA and IL-17RC, are abundantly expressed in CNS. However, whether IL-17A contributes to sympathetic excitation remains unknown.

Hypothesis:
IL-17A acts centrally to influence sympathetic drive and cardiovascular dynamics.

Methods:
BP (mmHg), HR (bpm) and RSNA (% change) were recorded in urethane anesthetized male Sprague Dawley rats. IL-17A was administered by intravenous (IV, 500 ng) or intracerebroventricular (ICV, 100 ng) injection or by bilateral microinjection (25 ng/side) into hypothalamic paraventricular nucleus (PVN). IL-17RA expression in the PVN was visualized by confocal microscopy.

Results:
IV IL-17A (n=6) induced a substantial and long-lasting increase (*p<0.001 vs. baseline) in BP (16.7 ± 2.1*), HR (57 ± 8*) and RSNA (104.5 ± 4.8 %*), that began within 45-60 mins and peaked 5-6 hours after IV injection. Similarly, ICV (n=6) or PVN microinjection of IL-17A (n=6) elicited dramatic and long-lasting increases in BP (23.1 ± 2.5 and 19.6 ± 2.1, respectively), HR (78.6 ± 7.4 and 65.1 ± 6.8, respectively) and RSNA (223.6 ± 5.1 and 152.2 ± 3.3, respectively), with the peak responses at 5 to 6 hours or even later. Confocal images showed that IL-17RA was richly expressed in the PVN.

Conclusion:
These data demonstrate that IL-17A regulates cardiovascular function and sympathetic drive, suggesting that IL-17A may contribute to sympathetic excitation in chronic inflammatory conditions like HTN and HF via its actions within the brain. IL-17A is a potential target for therapeutic intervention in these diseases.
Abstract 023: γδ T Cells Mediate αβ T Cell Activation in Angiotensin II-Induced Hypertension
Authors: Caillon Antoine , Paradis Pierre , Schiffrin Ernesto L
Published: September 2019

Objectives:
Both innate and adaptive immune cells have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γδ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69
+
) of αβ and γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension.

Methods:
C57BL/6 male mice were infused with Ang II (490 ng/kg/min, SC) for 7 and 14 days (n=5-7). All mice were 14-15 week-old at the end of the study. Spleen T cell profile was determined by flow cytometry. γδ and αβ T cells were isolated using magnetic beads from peripheral lymph nodes and spleen from C57BL/6 male mice treated or not with Ang II for 14 days. αβ T cells were cultured alone or with γδ T cells (5:1) in presence of anti-CD3 antibodies plus or minus Ang II, and αβ T cell phenotype was evaluated by flow cytometry.

Results:
Close correlations were demonstrated between the number (#) of activated CD69
+
γδ T cells and CD4
+
CD69
+
T cells (r
2
=0.74,
P
<0.01) and CD8
+
CD69
+
T cells (r
2
=0.64,
P
<0.01) after 7-day Ang II infusion. Correlations were also shown between the # of CD27
+
CD69
+
γδ T cells and CD4
+
CD69
+
T cells (r
2
=0.76,
P
<0.001) and CD8
+
CD69
+
T cells (r
2
=0.65,
P
<0.01) after 7-day Ang II infusion.
In vitro
, Ang II increased the fraction of CD69
+
αβ T cells when αβ T cells were co-cultured with γδ T cells isolated from control mice (% of αβ T cells: 19.9±2.9 vs. 16.4±2.6,
P
<0.001) but not when cultured alone. The fractions of CD69
+
(% of CD69
+
αβ T cells: 35.7±2.7 vs. 18.9±1.5,
P
<0.001) and CCR6
+
αβ T cells (% of CCR6
+
αβ T cells: 27.6±2.4 vs. 19.2±3.5,
P
<0.05) were increased when αβ T cells were co-cultured with γδ T cells isolated from Ang II-infused compared to control mice.

Conclusions:
These results suggest that γδ T cells mediate activation of αβ T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce the low-grade inflammation found in hypertension.
Abstract 030: Low Indoleamine 2,3 Dioxygenase Activity in Pregnancy are Associated With Increased Carotid-Femoral Pulse Wave Velocity in Preeclampsia
Authors: Santillan Mark K , Meincke Casee R , Scroggins Sabrina M , Betz Alexandria M , Brandt Debra S , Stroud Amy K , Santillan Donna A , Pierce Gary L
Published: September 2019

Preeclampsia is a prevalent multi-organ complication of pregnancy that is characterized by gestational hypertension and proteinuria. Immunologic and vascular alterations clearly occur before clinical disease. One mechanism that could explain both the exaggerated immune response and vascular dysfunction involves indoleamine 2, 3 dioxygenase (IDO). We have previously shown that the IDO-KO mouse replicates the vascular and obstetric phenotypes of human preeclampsia. We hypothesize that decreases in IDO activity are associated with vascular dysfunction as measured by carotid-femoral pulse wave velocity (PWV), a measure of aortic stiffness. We test this hypothesis in this nested case control study of two similarly recruited prospective cohorts (Preeclampsia IDO Vascular Function and Perinatal Outcomes study IRB: 201009701), and the (Early Vascular Dysfunction and Elevated Copeptin in Human Preeclampsia study IRB: 201503789). A total of 136 control and 13 preeclamptic pregnant women were recruited to measure vascular function and immunovascular factors throughout gestation. Plasma IDO activity was colorimetrically measured and trimester specific quartiles for IDO activity were calculated. PWV was measured via tonometry. Highest and lowest IDO activity quartile groups were compared. First trimester IDO activity was not associated with differences in PWV throughout gestation. Lower second and third trimester IDO activity was associated with higher PWV throughout gestation (1
st
trimester: 6.8 ± 0.3 vs 5.5 ± 0.2, P<0.001; 2
nd
trimester: 7.1 ± 0.4 vs 5.1 ± 0.2 P<0.001; 3
rd
trimester 6.5 ± 0.2 vs 5.9 ± 0.2 m/s, P<0.001). Even after controlling for preeclampsia, third trimester IDO activity was inversely associated with PWV (Beta IDO=-11, p=0.023 and Beta preeclampsia=0.150, p=0.753). Consistent with our hypothesis, our data demonstrate IDO activity is inversely associated with vascular dysfunction. Future studies will elucidate the molecular immunovascular mechanisms underlying this association in preeclampsia.
Abstract 033: Genetic Polymorphisms Associated With Apparent Treatment-Resistant Hypertension in Patients With Chronic Kidney Disease
Authors: Wang Ling , Pezeshkian Kevin , Wang Donna
Published: September 2019

The risk of suffering from apparent treatment-resistant hypertension (ATRH) increases more than 4-folds in patients with chronic kidney disease (CKD) comparing to general population, although causes unknown. We hypothesize that genetic variants of certain inflammatory factors occur in CKD patients with ATRH, which may serve as distinct genetic signature for ATRH. Data from Chronic Renal Insufficiency Cohort (CRIC) study were used to identify genetic variants related to ATRH among CKD patients. ATRH was defined as mean SBP/DBP ≥140/90 mm Hg while taking ≥3 antihypertensive medications or <140/90 mm Hg while taking ≥4 antihypertensive medications at the baseline visit. Clean genotype data on chromosome 6 from genotyped or imputed single-nucleotide polymorphisms (SNPs) were available for 3,263 patients (1,172 with ATRH and 2,091 without ATRH) in CRIC. Logistic regressions were carried out comparing distribution of SNPs among CKD patients with or without ATRH, controlling for confounding factors. Among 63,517 SNPs on chromosome 6, we found SNPs on three genes that were putative risk genes for ATRH among CKD patients (P-value<5х10
-4
). PHACTR1 (rs6900427) encodes for phosphatase and actin regulator 1, which plays a role in endothelial cell survival, and its variants have been associated with myocardial infarction and coronary artery disease. PPARD (rs6915115) encodes for peroxisome proliferator activated receptor delta, which has been implicated as an important inflammatory regulator including a role in the NF-κB pathway. Finally, PRKN (rs10945756) encodes for parkin RBR E3 ubiquitin protein ligase, which plays a role in mediating degradation of proteins including tumor necrosis factor receptor-associated factor 3 (TRAF3) that has been suggested to participate in the control of inflammation. Thus, these results are the first demonstrating that distinct genetic polymorphisms present in CKD patients with ATRH, which may serve as unique genetic signature for predisposed risk of ATRH. Moreover, association between predisposition of inflammatory factors and ATRH may implicate a role of inflammation in ATRH pathogenesis, revealing potential therapeutic targets.
Abstract 050: Essential Role of Cerebral Endothelial AT1 Receptors in the Blood-Brain Barrier Disruption Induced by Angiotensin-II Hypertension
Authors: Santisteban Monica M , Ahn Sung Ji , Lane Diane , Faraco Giuseppe , Garcia-Bonilla Lidia , Racchumi Gianfranco , Segarra Steven , Schaeffer Samantha , Poon Carrie , Shahanoor Ziasmin , Cipollini Virginia , Anrather Josef , Iadecola Costantino
Published: September 2019

The blood-brain barrier (BBB) controls the molecular exchange between blood and brain and is critical for maintaining brain health. Although hypertension (HTN) is well known to disrupt the BBB, the underlying cellular bases and the susceptible cerebrovascular segments(s) remain unclear. Here, we addressed these open questions in male mice in which HTN was induced by angiotensin II (Ang II; 600ng/kg/min s.c.; n=9-14/group) for 14 days. HTN increased BBB permeability to 3000 MW FITC-dextran, measured spectrophotometrically in brain extracts (30 ± 1 vs 18 ± 0.5 ng/g in controls; p<0.01).
In vivo
2-photon microscopy revealed that dextran leaks out more from microvessels >10ϒm (+38%; p<0.05) than cerebral capillaries (+7%; p>0.05). Electron microscopy showed that HTN induces tight junction remodeling (length -25%; complexity: -11%), and increases endothelial transcytosis (capillaries 1.9 and arterioles 3.5 folds; p<0.05), which is consistent with the downregulation of tight junction proteins (occludin: -17%; claudin-5: -30%) and of the transcytosis inhibitor Msfd2a (-43%). Since AT1 receptors (AT1R) on brain perivascular macrophages (PVM) mediate the deleterious cerebrovascular effects of Ang II HTN (JCI 126:4674), we tested if PVM also mediate the BBB dysfunction. However, PVM depletion (icv clodronate) or deletion of AT1R on PVM (bone marrow chimeras), attenuated the BBB dysfunction only partially (-60% and -49%, respectively; p<0.05), whereas AT1R-/- mice harboring AT1R+ PVM were completely protected (19 ± 1 ng/g; p>0.05 vs control), pointing to a key role of endothelial AT1R. Consistent with this prediction, cerebral endothelial AT1R deletion using a cerebral endothelial specific AAV-BR1-iCre in AT1R floxed mice prevented the BBB disruption completely (21 ± 1 ng/g; p>0.05 vs control). We conclude that AT1 signaling in cerebral endothelial cells initiates the BBB opening induced by Ang II HTN by increasing both paracellular and vesicular transport mainly in arterioles and venules, and that AT1 signaling in PVM, presumably from circulating Ang II crossing the BBB, amplifies the dysfunction. Such increase in BBB permeability to circulating agents may contribute to the cerebrovascular and cognitive dysfunction associated with HTN.
Abstract 055: Sex Differences in the Development of Age-Dependent Hypertension and the Salt Sensitivity of Blood Pressure
Authors: Puleo Franco J , Frame Alissa A , Chaudhary Parul , Wainford Richard D
Published: September 2019

Aim:
Age-dependent hypertension (HTN) develops at different rates in men versus women. These studies tested the hypothesis that in a model of normal aging, the Sprague Dawley rat, alterations in fluid and electrolyte balance contribute to age-dependent HTN in a sex-dependent manner.

Methods:
Three, 8 and 16-month old male and female SD rats on a normal salt (NS; 0.6% NaCl) diet underwent an acute IV volume expansion (VE; 5% body weight) and mean arterial pressure (MAP), diuresis and natriuresis were assessed. In separate groups of aged rats fed a 21-day NS or high salt (HS; 4% NaCl) diet in vivo MAP and NCC activity (ΔUNaV to IV hydrochlorothiazide, 2mg/kg) were assessed. N=4-6/gp.

Results:
The diuretic and natriuretic responses to an acute VE are profoundly blunted in aged male, but not female, rats. In contrast to female rats, male rats exhibit age-dependent HTN on a lifelong NS intake and develop age-dependent salt sensitivity of blood pressure. Female rats exhibit higher baseline sodium chloride cotransporter (NCC) activity and both young male and female rats exhibit dietary sodium evoked suppression of the NCC that is lost with age.

Conclusion:
Aging in the Sprague Dawley rat is associated with the sex specific development of age dependent HTN and the salt sensitivity of blood pressure in the male vs. female rat. We speculate that this sex specific difference is related to the impaired ability of male rats to maintain fluid and electrolyte homeostasis - potentially via a mechanism involving increased age-dependent baseline NCC-mediated sodium retention.
Abstract 057: Epigenome Wide Association Study Identifies DNA Methylation Sites Associated With Target Organ Damage in Elderly African Americans
Authors: Ammous Farah , Zhao Wei , Ratliff Scott M , Mosley Thomas H , Kardia Sharon L , Zhou Xiang , Smith Jennifer A
Published: September 2019

Introduction:
Epigenetic mechanisms, including DNA methylation, influence gene expression and mediate responses to the environment over the lifecourse. Previous research has identified a number of DNA methylation sites (CpGs) associated with arteriosclerosis and its risk factors. Given the underlying role of immunological and inflammatory processes in arteriosclerosis and the potential of pleiotropic effects of individual CpG sites across organ systems influenced by arteriosclerosis, we investigated the DNA methylation profiles of peripheral blood leukocytes from a cohort of African American (AA) hypertensive sibships to explore their association with measures of target organ damage (TOD), analyzed using both single and multi-trait (multivariate) models.

Methods:
Using the Genome-wide Efficient Mixed Model Association algorithm (GEMMA), we assessed the association between ~790,000 CpGs across the genome and 4 TOD traits: Estimated glomerular filtration rate (eGFR), urinary albumin-creatinine ratio (UACR), left ventricular mass index (LVMI) and relative wall thickness (RWT) in 961 subjects with mean baseline age of 57.5 years (SD: 10.3). DNA methylation was measured at baseline using the Infinium MethylationEPIC BeadChip and TOD was assessed 5 years later.

Results:
Single trait models adjusting for age, sex, blood cell proportions, genetic principal components, and time difference between the measures identified a single CpG site associated with UACR (cg04816311), LVMI (cg21134922) and RWT (cg03042953, all FDR < 0.1). Pleiotropic (multivariate) models identified 8 CpGs associated with at least one of the traits in the
CATSPERD, C7orf50, ALDH1L1, IFT43
and
OAT
genes. After adjusting for SBP, DBP and antihypertensive medication use, 6 CpGs remained significant and 1 additional CpG (cg21447579 in
SNORD116
gene cluster) was identified. After further adjustment for BMI, T2DM and smoking, two CpGs (cg02264946 in
CATSPERD
and cg12661888 in
IFT43)
remained significant.

Conclusions:
The study highlights the role of specific epigenetic sites in risk of TOD in an African American population. Additionally, it highlights the utility of using a pleiotropy-based analysis to identify CpG sites by leveraging information across related traits.
Abstract 065: Endothelial CULLIN3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension
Authors: Wu Jing , Fang Shi , Hu Chunyan , Otanwa Adokole J. J , Nair Anand R , Agbor Larry N , Lu Ko-Ting , Mukohda Masashi , Sigmund Curt D
Published: September 2019

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9) cause human hypertension (HT), which is likely to be driven by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 mutant causes vascular dysfunction and HT via augmented RhoA/Rho-kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3
in vivo
, we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE
ERT2
mice. The resultant mice (E-Cul3Δ9) developed nocturnal hypertension (Night time peak systolic BP, E-Cul3Δ9: 138±3 vs Control: 121±4 mmHg, p<0.01) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, p<0.01). No difference was seen in daytime BP. Nitric oxide synthase (NOS) inhibitor L-NAME induced smaller increases in nocturnal peak SBP and DBP in E-Cul3Δ9 mice (15±1 vs 27±3 mmHg, p<0.01), suggesting their NOS activity is low. Of note, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%, p<0.05) and cerebral resistance basilar artery (41% vs 77%, p<0.01). No difference in smooth muscle function was observed. To determine the molecular mechanisms, we isolated primary aortic endothelial cells from mice carrying the inducible Cul3Δ9 construct and induced Cul3Δ9 expression
in vitro
using adenovirus carrying Cre recombinase gene. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin3/PP2A/phospho-eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin-3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.
Abstract 068: Interplay Between Nox5 and Endoplasmic Reticulum Stress Regulates Vascular Signalling in Human Hypertension
Authors: Camargo Livia L , Montezano Augusto C , Wang Yu , Hussain Misbah , Awan Fazli R , Rios Francisco J , Touyz Rhian M
Published: September 2019

Nox5, a major ROS-generating oxidase in human vessels, regulates vascular contraction. We demonstrated an ER-perinuclear Nox5 localization and questioned the role of ER stress in Nox5 regulation. Vascular smooth muscle cells (VSMC) were isolated from small arteries from subcutaneous fat from normotensive (NT) and hypertensive (HT) subjects. Nox5 compartmentalization (cell fractionation); ROS generation (chemiluminescence); peroxiredoxin/DJ-1 oxidation, activation of ER stress and contractile signalling (IRE1α, Src, PKC, MLC phosphorylation; immunoblotting) and actin cytoskeleton organization (phalloidin staining) were assessed. In hypertension, ROS levels (139±27% vs NT, p<0.05), oxidation of peroxiredoxin (870.4±188.7% vs NT, p<0.05) and DJ-1 (125±34% vs NT, p<0.05) were increased. IRE1α phosphorylation was increased in the HT group (58±21% vs NT, p<0.05). ER stress inhibition (4-PBA, 1mM) reduced ROS levels in HT subjects (20±6% vs NT, p<0.05), suggesting association between ER and oxidative stress. Nox5 expression was increased in the HT group (103±23% vs NT, p<0.05) in a compartment specific manner: Nox5 levels were reduced in plasma membrane (45±7% vs NT, p<0.05), but increased in the ER/nuclear fraction (46±13% vs NT, p<0.05). IRE1 inhibition (STF083010, 60μM) decreased Nox5 expression in the HT group (65±5% vs Ctl, p<0.05), while induction of ER stress (tunicamycin, 5μg/ml, 24h) increased Nox5 expression in cells from both groups (p<0.05). To investigate the role of Nox5 on contractile signalling, cells were treated with mellitin (100nM), a Nox5 inhibitor. ROS generation and phosphorylation of c-Src, PKC and MLC
20
induced by Ang II were reduced by mellitin in both groups (p<0.05 vs Ctl). In contrast, silencing of p22phox increased ROS and activation of c-Src, PKC and MLC
20
in both groups, an effect blocked by mellitin (p<0.05 vs Ctl). VSMC from hypertensive subjects had increased number of stress fibres, an effect attenuated by mellitin. Our findings demonstrate that Nox5 is upregulated in a compartment specific manner and is regulated by ER stress in hypertension. Nox5 upregulation influences pro-contractile signalling and cytoskeleton reorganization in VSMC, processes that contribute to vascular dysfunction in hypertension.
Abstract 074: Vascular Dysfunction in Boys With Androgen Insufficiency - Role of Nox5 and Calcium
Authors: Lucas-Herald Angela , Alves-Lopes Rheure , Haddow Laura , O'Toole Stuart , Amjad S Basith , Flett Martyn , Lee Boma , Steven Mairi , Pashova Antoniya , Montezano Augusto , Ahmed S Faisal , Touyz Rhian
Published: September 2019

Hypospadias is usually associated with androgen insufficiency. As androgens regulate Ca
2+
influx, Nox activity and redox signalling, we sought to ascertain where these may be implicated in vascular dysfunction seen in boys with hypospadias. Peripheral arteries dissected from excess foreskin tissue from hypospadias (cases) or circumcision (controls) were used. Vascular function was assessed by wire myography.VSMCs were cultured and ROS production was measured by amplex red and chemiluminescence. mRNA expression was measured by qPCR and Ca
2+
influx by fluorescence microscopy. 20 boys with hypospadias and 29 age-matched controls were enrolled in this study (median age 1.9 (range 1.3, 12.2) years). Arteries from cases demonstrated increased constriction to U46619 vs controls (Emax %KCl: 175.6 vs 66.3 p<0.05). Expression of Nox5 was increased in cases (2.6 fold,p<0.05), while a decrease in Nox4 expression was observed (9.5 fold, p<0.05). VSMC superoxide anion (5.3 fold) production and H
2
0
2
(3.3 fold) levels were increased in cases compared to controls (p<0.05), an effect inhibited by melittin. Melittin, a Nox5 inhibitor, inhibited contraction in arteries from boys with hypospadias (Emax %KCl: 137.9 vs 62.9, p<0.05). There was no difference in mRNA expression of androgen receptors (
AR
and
GPRC6A)
between cases and controls but cases had higher expression of the oestrogen receptors,
ESR1
(2.7 fold),
ESR2
(2.6 fold) and
GPR30
(2.9 fold) (p<0.05).Boys with hypospadias demonstrated increased CACNA1C (3.9 fold), SERCA (3.1 fold), TRPM2 (2.0 fold) and Ryr1 (3.5 fold) mRNA expression (p<0.05).There was no basal difference between Ca
2+
influx between controls and boys with hypospadias.Testosterone and oestradiol stimulation reduced expression of Ca
2+
channels (p<0.05). Moreover, testosterone (p=0.04) and oestradiol (p=0.02) reduced Ca
2+
influx in boys with hypospadias. Vascular dysfunction observed in small arteries from boys with hypospadias may be due to Nox5 activation and altered Ca
2+
regulation. These data suggest a role for androgens in the regulation of vascular Nox5-Ca
2+
signaling, which when perturbed may predispose to cardiovascular risk.
Abstract 075: Sex Differences in the Development of Autoimmune-Associated Hypertension
Authors: Mathis Keisa W , Young-Stubbs Cassandra , Morales Jessica
Published: September 2019

Systemic lupus erythematosus (SLE) is an autoimmune disorder with prevalent hypertension and is a novel disease model to study the link between chronic inflammation and hypertension. Because SLE is female-dominant, studies are usually conducted in female
NZBWF1
mice, a well-established model of SLE. However, men are also diagnosed with SLE and the late onset and different clinical features draws interest. We hypothesize that sex differences exist in SLE and that female SLE mice have heightened disease, hypertension, and renal injury earlier than males. Female and male SLE and control (
NZW
) mice were monitored for albuminuria starting at 30 weeks of age and then at 35 weeks implanted with vascular catheters to allow measurement of blood pressure in conscious mice. At 30 weeks, female SLE mice had elevated plasma dsDNA autoantibodies, the hallmark of SLE, compared to male SLE (4.6e5 ± 1.3e5 vs. 6.3e4 ± 2.7e4 U/mL; n=3-5; all p<0.05), female controls (8.9e4 ± 3.3e4), and male controls (4.8e4 ± 9.3e3). At this same time point, 32% (7 out of 22) of female SLE mice had albuminuria versus 5% (1 out of 20) of male SLE, 5% (1 out of 22) of female controls, and no male controls. Although male SLE mice did not reach similar levels of dsDNA autoantibodies as SLE females (2.0e5 ± 4.6e4 vs. 3.9e5 ± 4.3e4; n=8-13; p=0.015), by 35 weeks, the autoantibodies were increasing in SLE males (2.0e5 ± 4.6e4 vs. 6.3e4 ± 2.7e4; p=0.157) and higher than male controls (4.3e4 ± 9.3e3; p=0.046). At 35 weeks, 63% (10 out of 16) of female SLE mice had albuminuria versus 5% (1 out of 19) of male SLE, 5% (1 out of 22) of female controls, and no male controls. Importantly, both female and male SLE mice were hypertensive at 35 weeks: blood pressure was higher in female SLE than female controls (152 ± 5 vs. 126 ± 3 mmHg; n=6-8; p=0.003) and in male SLE compared to male controls (152 ± 4 vs. 136 ± 4 mmHg; n=6-11; p=0.041). These data confirm the development of hypertension and renal injury in female SLE mice and suggest that male SLE mice develop hypertension with no renal involvement. Future studies will allow us to investigate the divergent mechanisms involved in autoimmune-induced hypertension in female and male mice, which will ultimately provide clues in sex differences in hypertensive humans with underlying chronic inflammation.
Abstract 076: Sex Difference in the Effects of Dopamine D2 Receptor Specific Deletion in the Renal Proximal Tubules of Mice
Authors: Kumar Megha , Konkalmatt Prasad , Asico Laureano D , Hunt Jessica , Latham Patricia , Jose Pedro A , Armando Ines
Published: September 2019

Dopamine receptor D2 (D2R) plays a significant role in kidney function by maintaining normal blood pressure (BP) and preventing renal inflammation and injury. D2R silencing in the mouse kidney increases BP and increases renal injury. To study the effects of D2R in the renal proximal tubule we generated

Drd2
fl/fl
,

P

SGLT2
::Cre+

mice (D2R
PT-/-
) that lack D2R only in the renal proximal tubule and

Drd2
fl/fl
,

P

SGLT2
::Cre-

mice (D2R
PT+/+
) that do not have the deletion. We studied male and female mice on normal salt (NS; 0.4% NaCl) and high salt (HS; 4% NaCl) diets. Mice were genotyped for

Drd2
fl/fl

and a smaller amplicon representing the Cre deletion mutant. On NS diet, male D2R
PT-/-
had higher systolic BP (SBP) measured under anesthesia than male D2R
PT+/+
(113±1 vs 102±3 mmHg, n=5/group; P<0.05) and female D2R
PT-/-
(104±5 mmHg) or female D2R
PT+/+
(106±3 mmHg). SBPs on HS diet were similar in D2R
PT-/-
females and males. On NS diet renal mRNA expressions of TNF-α, TGFβ1, Fn1, and Col1a1 were higher (P<0.01) in female D2R
PT+/+
than in male D2R
PT+/+
; conversely the expression of the kidney injury marker, Kim-1, was higher in male than female D2R
PT+/+
(P<0.01). Male D2R
PT-/-
expressed less (P<0.01) renal TNF-α, TGFβ1, Col1a1, cell proliferation marker Mki-67, and Fn1 than female D2R
PT-/-
. However, the renal expression of Kim-1 was less (P<0.01) in female than male D2R
PT-/-
. On HS diet, renal mRNA expressions of TNF-α, TGFβ1, and Fn-1 were similar in both male and female D2R
PT+/+
and D2R
PT-/-
. HS diet increased (P<0.05) the expression of Col1a1 and Kim-1 in male D2R
PT+/+
and D2R
PT-/-
but not in females of the two genotypes; HS diet also increased the expression of Mki-67 only in D2R
PT-/-
males. Our data show striking differences in the inflammatory response, cell proliferation, and kidney injury between males and females, with females expressing more inflammatory and proliferation markers but less injury markers than males. Deletion of the D2R in the proximal tubule increases the renal expression of extracellular matrix proteins and inflammatory and proliferation markers, effects that were more pronounced in females, except for Kim-1. The differences between males and females are maintained on HS diet, indicating that female mice have a “healthier” response to renal injury.
Abstract 086: Pvn-Specific Microgliosis Drives Inflammation in G-Alpha-i
2
Protein Dependent Salt Sensitive Hypertension and Gnai2 Snps Correlate With Essential Hypertension
Authors: Moreira Jesse D , Chaudhary Parul , Nist Kayla M , Abkin Eric , Frame Alissa A , Puleo Franco , Wainford Richard D
Published: September 2019

Aim:
Failure to upregulate hypothalamic paraventricular nucleus (PVN) Gαi
2
proteins promotes salt sensitive hypertension (ssHTN) and central inflammation plays a role in HTN. We hypothesize that 1) PVN-specific inflammation contributes to Gαi
2
protein dependent ssHTN, and 2) GNAI2 SNPs correlate with HTN.

Methods:
3-month-old male Sprague Dawley rats were implanted with I.C.V. cannulas fitted to osmotic minipumps and infused with Gαi
2
or control scrambled (SCR) oligodeoxynucleotides (ODN) (25μg/5μl/day), or ODN + minocycline (MINO) (120μg/5μl/day, HS only) and placed on a 7-day normal (NS; 0.6% NaCl) or high (HS; 4% NaCl) salt diet. Day 7 BP (femoral cannulation) and Plasma NE (ELISA) were measured. PVN and subfornical organ microglia and astrocyte activation (IHC), and PVN pro-inflammatory (PIC) and anti-inflammatory (AIC) cytokine mRNA and protein levels were analyzed. GNAI2 SNPs were examined in the UK BioBank for associations with HTN.

Results:
In contrast to SCR ODN infusion, Gαi
2
ODN infusion and HS evoked HTN, elevated plasma NE, PVN-specific microgliosis, upregulation of PVN PICs and downregulation of PVN IL-10. MINO co-infusion with Gαi
2
ODN and HS diet restored PIC and AIC mRNA and protein levels. Furthermore, MINO attenuated microgliosis and reduced the magnitude of ssHTN. GNAI2 SNPs rs2298952 (p=0.0411, MAF=0.103) and rs4547694 (p=0.0117, MAF=0.381) are both associated with essential HTN in BioBank subjects.

Conclusion:
PVN-specific microgliosis and inflammation may drive Gαi
2
protein dependent ssHTN and GNAI2 SNPs represent a potential biomarker of essential HTN.
Abstract 093: Novel Mir-21-5p-/- Rat Model Exhibits Protection Against Chronic Kidney Disease Associated Cardiac Dysfunction
Authors: Nasci Victoria L , Geurts Aron , Kriegel Alison J
Published: September 2019

We have used a surgical model of CKD, the 5/6 nephrectomy (5/6 NX) to study molecular mechanisms involved the development of cardiac pathology in Sprague Dawley (SD) rats, identifying elevated myocardial miR-21-5p as a regulator of metabolic dysfunction with the use of a miR-21-5p inhibitor. To study this regulatory mechanism further we sought to develop an animal model that lacks miR-21-5p expression. Here we introduce the first reported line of miR-21 null mutant rats, generated on a Sprague Dawley background using CRISPR gene editing technology. Sequencing shows a deletion of nucleotides 4-13 of the primary miR-21 sequence. This line was maintained as an outbred line by continuous backcross to vendor SD at each generation. Heterozygous offspring were crossed to generate miR-21-/- and wild-type (WT) control littermates. Analysis by qRT-PCR indicates a global absence of miR-21-5p and miR-21-3p in miR-21-/- rats. The objectives of this study were 1) to examine the effect of the loss of both miR-21-5p and miR-21-3p on cardiac phenotypes in the 5/6Nx model, and 2) to evaluate these phenotypes in male and female rats. We performed echocardiography on 10 week-old male and female WT and miR-21-/- and then randomly assigned them to undergo sham or 5/6Nx. Echocardiography, acute arterial blood pressure and pressure-volume analysis was performed on anesthetized rats 7 weeks later. Our data indicate that male and female miR-21-/- rats are protected from developing LV dysfunction and hypertrophy following 5/6Nx surgery. We observed no statistical difference in anesthetized blood pressures within groups of either sex, though pressures tended to be elevated in 5/6Nx rats regardless of genotype. Among several indices of improved cardiac function observed in miR-21-/- rats was an enhanced fractional shortening in both males (WT 40.89 ± 1.70% vs. miR-21-/- 50.10 ± 3.27%) and females (WT 44.98 ± 1.96% vs. miR-21-/- 53.00 ± 7.37%) 7 weeks after 5/6Nx. These data indicate that the miR-21-/- exhibit a cardioprotective phenotype that recapitulates that observed with administration of miR-21-5p inhibitor, despite a concomitant loss of miR-21-3p. We conclude that this is an appropriate model for studying the role of miR-21-5p regulation CKD-associate cardiac dysfunction.
Abstract 102: Genetic Deletion of Sirtuin 3 (SIRT3) in the Mitochondria of the Proximal Tubules Aggravates Angiotensin II-Induced Hypertension by Impairing the Pressure-Natriuresis Response in Proximal Tubule-Specific SIRT3 Knockout Mice
Authors: Li Xiao C , Chen Xu , Zheng Xiaowen , Zhao Chunling , Chen Jian X , Zhuo Jia L
Published: September 2019

Sirtuin 3 (SIRT3), a key mitochondrial protein in the sirtuin family, plays a key role in maintaining normal mitochondrial function via its anti-oxidative, anti-aging, and anti-inflammatory effects. Conversely, angiotensin II (ANG II), a key peptide of the renin-angiotensin system, plays a key role in the development of cardiovascular and hypertensive injury through its vasoconstrictive, pro-oxidative, pro-growth, and proinflammatory actions, in part by suppressing mitochondrial SIRT3 expression and inducing mitochondrial dysfunction. The present study tested the hypothesis that genetic deletion of SIRT3 selectively in the proximal tubules of the kidney aggravates ANG II-induced hypertension in proximal tubule (PT)-specific SIRT3 knockout mice (PT-SIRT3-KO). PT-SIRT3-KO mice were generated using the SGLT2-Cre/SIRT3-loxP approach. ANG II-dependent hypertension was induced by infusing a slow pressor dose of ANG II, 0.5 mg/kg/day, i.p., and a 2% Na
+
diet for 2 weeks in adult male wildtype (WT) and PT-SIRT3-KO mice (n=7-12 per group). PT-SIRT3-KO mice developed normally, and showed no abnormal cardiac and kidney structures. Under basal conditions, systolic, diastolic and mean arterial pressure were significantly lower (WT: 119 ± 3 mmHg vs. PT-SIRT3-KO: 111 ± 3 mmHg,
P
<0.05), whereas urinary Na
+
excretion was significantly higher in PT-SIRT3-KO than WT mice (WT: 167.9 ± 5.8 μmol/24 h vs. PT-SIRT3-KO: 217.9 ± 7.9 μmol/24 h,
P
<0.05), without altering urinary K
+
excretion. ANG II infusion significantly increased systolic blood pressure by 24 ± 3 mmHg in WT mice (
P
<0.01), whereas it increased systolic blood pressure by 38 ± 3 mmHg in PT-SIRT3-KO mice (
P
<0.01). Conversely, ANG II-induced hypertension was associated with a marked natriuretic response in WT mice (547.7 ± 19.9 μmol/24 h), which was attenuated in PT-SIRT3-KO mice (312.1 ± 11.1 μmol/24 h,
P
<0.01). Proximal tubule-specific SIRT3 deletion slightly increased the kidney to body wt. ratio (n.s.), but significantly attenuated the heart to body wt. ratio in PT-SIRT3-KO mice (
P
<0.01). Our results suggest that genetic deletion of mitochondrial SIRT3 in the proximal tubules of the kidney aggravates ANG II-induced hypertension by impairing the pressure-natriuretic response in PT-SIRT3-KO mice.