bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 212: Reduced Magnesium in the Vascular Microenvironment May Be the Switch That Increases Susceptibility to Vascular Calcification in Experimental Chronic Kidney Disease
Authors: Zelt Jason G , McCabe Kristin M , Shobeiri Navid , Maio-Twofoot Tina , Holden Rachel M , Adams Michael A
Published: March 2022

Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Pathogenic vascular calcification (VC) in CKD, likely resulting from altered phosphate and calcium metabolism, is a critical harbinger of CVD in these patients. A growing body of evidence suggests a protective role for magnesium in attenuating VC. In this study, the objective was to elucidate the stoichiometry of mineral accumulation in the vascular media, particularly with regard to the relative abundance of magnesium, phosphate and calcium, during the various stages of progression of VC in male Sprague Dawley rats (n=40) with adenine-induced CKD (0.25% in diet) for up to 7 weeks. The adenine protocol generated a range of CKD severity (creatinine, 234 to 650 μmol/L, mean 353 ± 91 μmol/L) and we further modified the susceptibility to VC by altering vitamin K status. Specifically, we increased vitamin K intake (K1 @ 100 mg/kg diet) or decreased availability (warfarin, ~0.1 mg/kg/day) to induce a decreased and increased VC, respectively. Calcium, phosphate, and magnesium contents were analyzed in the thoracic aorta, abdominal aorta and pudendal arteries. Accumulating tissue phosphate (~2.5 to 1100 nmol/mg) positively correlated with both tissue calcium and magnesium levels (r2=0.99, p<0.0001; r2=0.86, p<0.0001 respectively). However, stoichiometric analysis revealed the relative magnesium abundance decreased markedly with progressing VC (~20% to <2% Mg when PO4 greater than 30 mmol/mg tissue), indicating a trajectory away from the more soluble magnesium containing crystal, whitlockite. Our research suggests that perturbations in Mg homeostasis can mediate changes in the microenvironment that facilitate the progression of VC. Specifically, a relative decrease of magnesium within the vascular media could reduce the protective capacity and potentiate the calcification process.
Abstract 222: Impairment of Autonomic Function and Increased Aldosterone in Metabolic Syndrome Model Rats Mediates Circadian Blood Pressure Rhythms
Authors: Sueta Daisuke , Ogawa Hisao , Kim-Mitsuyama Shokei
Published: March 2022

[Aim]
Metabolic syndrome (MetS) is significantly associated with abnormal blood pressure (BP) circadian rhythms, although the underlying mechanism(s) is unclear. Using SHR/NDmcr-cp (SHRcp), manifesting with hypertension, obesity, insulin resistance and dyslipidemia, we examined the mechanisms underlying abnormal circadian rhythms of BP in MetS.

[Methods]
(1)We surgically implanted telemetry devices in control SHR and SHRcp to monitor BP continuously. We performed a cross-spectral analysis, using a fast Fourier transformation algorithm, of arterial pulse waves to determine autonomic function and baroreceptor reflex function. Urine samples in dark (active) phase and light (inactive) phase were collected with metabolic cages to measure factors associated with blood pressure control.

(2)SHRcp were orally administered azilsartan (1mg/kg/day), an AT1 receptor blocker, once daily at the beginning of the active phase, to examine the effects of azilsartan on the above mentioned parameters.

[Results]
(1)SHRcp exhibited non-dipper type hypertension, although SHR exhibited dipper-type hypertension. Compared with SHR, SHRcp showed an increased low frequency (LF) power of sBP and a decreased spontaneous baroreceptor reflex gain (sBRG). Thus, SHRcp was characterized by enhanced vascular sympathetic tone and impaired baroreflex function 24 hrs a day. Urine norepinephrine(NE) and aldosterone excretion levels were greater in SHRcp over 24 hrs than in SHR.

(2)Azilsartan treatment once daily normalized BP in SHRcp over 24 hrs. Azilsartan had a greater hypotensive effect in SHRcp than in SHR, thus indicating the key role of hypertension in SHRcp. Azilsartan decreased LF of sBP, increased sBRG, and reduced urinary NE excretion levels in SHRcp. Azilsartan also reduced urinary aldosterone excretion over 24 hrs and serum aldosterone concentration, thus increasing urinary sodium excretion in SHRcp.

[Conclusion]
SHRcp, a model of MetS,exhibit non-dipper type hypertension, and thus they are suitable models for studying abnormal BP circadian rhythms. The mechanism of abnormal BP circadian rhythm in SHRcp was attributed to an impaired autonomic nervous system and aldosterone-induced sodium retention, which were both mediated by AT1 receptor.
Abstract 226: Importance of Measuring the Time Course of Flow Mediated Dilation When Assessing Endothelial Function in Obese African Americans
Authors: Celedonio Jorge E , Mariños Alejandro , Ramirez Claudia E , Gottlieb JoAnn , Gamboa Alfredo , Biaggioni Italo , Shibao Cyndya
Published: March 2022

Flow mediated dilation (FMD) is a noninvasive technique to assess endothelial function in humans. In most studies FMD is reported as the percent change in brachial artery diameter at 60 seconds after cuff deflation relative to baseline (%FMD at 60 seconds). Recently the availability of automated computer-based edge detection allows measurement of peak dilation during the entire time course of brachial dilation post-hyperemia. Considering that changes in arterial diameter in response to shear stress is a continuous process and that peak dilation may differ in pathological states and occur at any arbitrary time point, we examined the time course of brachial artery FMD after a 5-min period of ischemia in 38 obese African American women (42±9.3 years old, BMI 39±6, % body fat 48.2±4.3, SBP 131±13, DBP 83±8 mm Hg, HR 77±12 bpm). Results were analyzed using brachial analyzer software (Medical Imaging Applications LLC). Baseline brachial artery diameter was 3.95±0.55 mm, the time to peak was 48.93 ±13.82 seconds. The percent change in FMD (%FMD) obtained at peak diameter was greater (4.7±3.19 mm) than %FMD at 60 seconds (3.23±4.33 mm); mean difference = 1.4 (95% CI, 0.54-2.4, P=0.003). A large proportion (79%) of true peak diameters fell outside the 60 seconds time frame typically used to assess FMD, Figure. Our results suggest that the peak vasodilator effect occurs before 60±5 seconds in the majority of our population. Therefore, the evaluation of FMD as a continuous measurement has greater accuracy and less variability for the evaluation of FMD compared with an arbitrary time point.
Abstract 232: High Glucose Attenuates In Vitro Tube Growth of Microvascular Endothelial Cells
Authors: Prisco Anthony R , Hoffmann Brian R , Bukowy John D , Karcher Jamie R , Greene Andrew S
Published: March 2022

Aim:
To investigate how high glucose affects the ability of microvascular endothelial cells to form tube-like structures
in vitro
and to develop open source software that automates the analysis of tube formation assays.

Background:
Over 300 million people worldwide suffer from diabetes. Patients with diabetes experience impaired angiogenesis. Tube formation assays (TFAs) mimic
in vivo
angiogenesis. During a TFA, endothelial cells that are placed onto a matrigel coated surface arrange into tube-like structures. Quantification of TFAs is difficult; automation typically requires a fluorescent stain and a specific software package. Manual quantification is tedious and subject to inter-user variability. In this study, we describe how high glucose affects the ability of rat microvascular endothelial cells (RMVECs) to form tube-like structures and the development of an open source tool called “Pipeline” that analyzes brightfield TFA images quantifying the total tube length and number of branchpoints.

Methods:
RMVECs were cultured in media with normal (5.6 mM) or high glucose (25 mM) for one and two week intervals. RMVECs were added to four-chamber slides coated with growth factor reduced Matrigel. After 24 and 48 hours, brightfield images were taken of the TFA. Images were analyzed by manually tracing the total tube length in MetaMorph image processing software or automatically using Pipeline. Results from automatic and manual analysis were compared. The algorithm was validated by measuring the total length of images generated
in silico
. The code was compiled in C and will run on any computer with the freely available MATLAB R2012a runtime environment.

Results:
A significant decrease in the total tube length was found when comparing groups treated with high glucose for 2 weeks versus 1 weeks (20.3 +/- 1.4mm vs 25.2 +/- 1.3mm, p<0.05). Additionally, cells in high glucose for 2 weeks versus 1 week were found to have significantly less branch points (68 +/- 6.1 vs 90.9 +/- 5.8, p<0.05). When validating the Pipeline algorithm against images generated
in silico
(at multiple orientations), Pipeline was able to detect total image length within 1% (9216μm vs 9348 +/- 98μm). Analysis using pipeline was found to be 25X faster than manual analysis (7 minutes versus 180 minutes).
Abstract 24: Tubular Vegfa is Required for Renal Microvasculature and Oxygen Sensing
Authors: Dimke Henrik , Sparks Matthew A , Frische Sebastian , Coffman Thomas M , Quaggin Susan E
Published: March 2022

Adequate renal oxygenation is pivotal for maintaining essential functions of the kidney. Vascular endothelial growth factor A (VEGFA) plays a key role in vascular formation and maintenance, processes that are critical for sufficient tissue perfusion and oxygenation. Here we show that
Vegfa
is expressed in select tubular epithelial cells within the nephron, while expression of its receptor (
Kdr/Vegfr2
) is restricted largely to capillary beds. Early genetic ablation of tubular
Vegfa
allows the formation of a grossly normal kidney. However, microvascular density is markedly reduced, leading to increased renal hypoxia. As a consequence the mice develop pronounced polycythemia (81 ± 1.62
vs.
58 ± 0.87 % Hematocrit), due to an increase in serum erythropoietin concentration (426.5 ± 49.3
vs.
249.3 ± 13.6 pg/ml). The elevation in circulating erythropoietin is elicited by an augmented renal production of the hormone, and occurs independent of changes in hepatic expression. In kidney, expression of classical hypoxia-inducible factor-1 responsive transcripts remains unaltered; suggesting that erythropoiesis partly compensates the capillary rarefaction. An increase in diastolic blood pressure (DBP, 112 ± 3.7
vs.
104 ± 1.6 mmHg) is present in mice lacking tubular Vegfa maintained on a normal diet, while systolic blood pressure (SBP, 139 ± 2.8
vs.
135 ± 2.8 mmHg) does not change. The difference in diastolic pressure is ablated when dietary NaCl content changed (DBP: 107 ± 3.1
vs.
103 ± 2.7 mmHg; SBP: 132 ± 4.1
vs.
131 ± 2.9 mmHg; for low NaCl), or when angiotensin II is infused (DBP: 137 ± 6.7
vs.
134 ± 3.8 mmHg; SBP: 161 ± 7.6
vs.
162 ± 9.3 mmHg). Moreover, renal electrolyte excretion remains unperturbed by reductions in tubular Vegfa. In conclusion, tubular Vegfa is critical for adequate development of renal microvasuculature, a prerequisite for proper oxygenation of the kidney. Compensatory polycythemia likely prevents severe perturbations of renal function in this model. In addition, increased diastolic blood pressure may occur as a consequence of capillary rarefaction within the kidney.
Abstract 241: Splenic T Regulatory Lymphocytes Are Reduced in Genetically Hypertensive Rats Associated with Central Sympathoexcitation
Authors: Katsuki Masato , Hirooka Yoshitaka , Kishi Takuya , Sunagawa Kenji
Published: March 2022

Background:
Chronic inflammation plays a role in the pathogenesis of hypertension. Among the subsets of T lymphocytes, T regulatory lymphocytes (Tregs), which are immune suppressors of inflammatory responses, are reported to prevent angiotensin II (AngII)-induced hypertension and vascular injury. We hypothesized that Tregs play a critical role in the development and maintenance of hypertension. The aim of the present study was to determine whether Tregs are decreased in genetically hypertensive rats at both prehypertensive and established ages compared with those in normotensive rats, and if so, to examine whether blockade of brain AngII type 1 receptors (AT1R) affects Tregs in hypertensive rats.

Methods and Results:
We examined Tregs in stroke-prone spontaneously hypertensive rats (SHRSP) at prehypertensive (5 week old) and established ages (12 week old) and age-matched normotensive Wistar-Kyoto rats (WKY). In both SHRSP and WKY, the transcription factor forkhead box P3 (Foxp3) mRNA expression levels were greater in the spleen than in other organs. However, Foxp3 mRNA and protein expression levels in the spleen were significantly lower in SHRSP than in WKY (Foxp3 mRNA: 0.47±0.03 vs. 1.02±0.03, protein: 0.39±0.02 vs. 0.66±0.03, n=6 for each, p<0.05). Flow cytometric analysis revealed that CD4+CD25+Foxp3+ cells (Tregs) in the spleen were significantly lower in SHRSP than in WKY (%Foxp3+ gated on CD4+CD25+ cells in the spleen; 45.4±1.4 vs. 58.1±2.0, n=3 for each, p<0.05). In addition, CD4+CD25+Foxp3+ cells in the spleen were lower in prehypertensive SHRSP, but not in the thymus. In hypertensive SHRSP, intracerebroventricular infusion of AT1R blocker (losartan, 1mg/kg/day for 2 weeks) reduced blood pressure and 24-hour urinary norepinephrine excretion, and upregulated Foxp3 mRNA expression levels in the spleen (0.44±0.04 vs. 0.31±0.02, n=3-4, p<0.05, vs. vehicle-treated SHRSP).

Conclusion:
Our findings indicate that splenic Tregs are reduced in SHRSP at both prehypertensive and established hypertensive stages. These alterations may contribute to the development and maintenance of hypertension. Central sympathoinhibition increases the reduced splenic Tregs in SHRSP suggesting interaction between sympathetic hyperactivity and Tregs.
Abstract 247: Cardiovascular Risk in Black and White Children: The Influence of Parental Health Status
Authors: Staiano Amanda E , Katzmarzyk Peter T
Published: March 2022

Objective:
Parental cardiovascular disease (CVD) may indicate children’s CVD risk. This study examined whether parental obesity and disease were associated with child CVD risk factors, independent of child obesity, and whether associations differed by race.

Methods:
The sample included 395 White and Black 5-18 year-old children. Parental obesity was determined by self-reported height and weight, while their disease status was self-reported diagnosis of coronary heart disease (CHD), hypertension and hypercholesterolemia. Child’s risk factors included waist circumference (WC); resting systolic (SBP) and diastolic blood pressure (DBP) percentiles; and fasting triglycerides, HDL-C, LDL-C, total cholesterol, glucose and C-reactive protein (CRP). BMI z-scores (BMI-z) were calculated using CDC growth charts. Analysis of variance, unadjusted and adjusted for BMI-z, was used to examine associations of parental obesity and disease with each child risk factor, stratified by race.

Results:
Black children had higher BMI-z than White children (1.1 vs. 0.8, p<0.01) and were more likely to have parents with obesity (p<0.0001) and hypercholesterolemia (p<0.05), but not CHD or hypertension. Parental obesity was significantly associated with WC, SBP and DBP in White and Black children and with CRP in White children (all p<0.05). When adjusted for BMI-z, parental obesity was significantly associated with SBP in White children (p<0.05). Parental CHD was significantly associated with WC in Black children in both unadjusted and adjusted models (p<0.05), with CRP in Black children in unadjusted models (p<0.05), and with triglycerides in White children in adjusted models (p<0.05). Parental hypertension was associated with WC and triglycerides in White children in unadjusted models (p<0.05), and with no risk factors in adjusted models. Parental hypercholesterolemia was not associated with risk factors, except LDL-C in White children in adjusted models (p<0.05).

Conclusion:
Parental obesity and disease were associated with CVD risk factors in White and Black children, but child BMI-z attenuated many associations to non-significance. Further research is needed to investigate the additive utility of parental medical history to stratify children’s CVD risk.
Abstract 251: 20-HETE Contributes to the Hypertension in a Model of Postmenopausal Polycystic Ovary Syndrome
Authors: Patil Chetan , Moulana Mohadetheh , Romero Damian , Roman Richard J , Baker Rodney , Reckelhoff Jane F
Published: March 2022

Polycystic ovary syndrome (PCOS) is the most common reproductive disorder in premenopausal women. PCOS is characterized by hyperandrogenemia that does not abate after menopause. Hyperandrogenemic postmenopausal women (PMW) are hypertensive, but the mechanisms responsible have not been elucidated. Hypertension (HT) in PMW is less well controlled than in age-matched men, which suggests that different mechanisms may be mediating the HT in PMW. We have developed a model, the hyperandrogenemic postcycling female (PMHAF) rat, aged 13-14 mos, that exhibits many of the characteristics found in hyperandrogenemic PMW. In the present study we tested the hypothesis that 20-HETE contributes to the HT in PMHAF rats. Female Sprague-Dawley rats were implanted with dihydrotestosterone (7.5mg/90d) or placebo pellets beginning at 4 wks of age, and pellets were changed every 85 d. At 13-14 mos of age, preglomerular renal microvessels were isolated from PMHAF and placebo control rats (n=6/grp) and 20-HETE and ω-hydroxylase activity were measured by LC-MS. mRNA for CYP4A enzymes were measured by real time RT-PCR. Basal 20-HETE levels in the renal vasculature increased by 60% in PMHAF vs placebo controls (0.398±0.22 vs 0.245 ±0.68 pmol/mg; p<0.05) and 20-HETE synthesis from arachidonic acid was increased by 17-fold (19.6±5.1 vs 1.18±0.14 pmol/min/mg, p<0.001). CYP4A2 mRNA levels were increased 15-fold and CYP4A8 mRNA levels were decreased by 60% in PMHAF rats compared to placebo controls (n=6/grp; p<0.05, PMHAF compared to placebo controls). In another group of rats, acute infusion of an inhibitor of 20-HETE synthesis, HET-0016 (10 mg/kg bolus and 1 mg/kg/h iv) reduced MAP by about 20 mm Hg in PMHAF rats. These data suggest that synthesis of 20-HETE in the vasculature is upregulated in PMHAF rats and contributes to their hypertension. The studies also suggest that novel mechanisms may be responsible for hypertension in PMW with hyperandrogenemia compared to age-matched men, and as such, novel therapeutic approaches may be necessary to control their blood pressures. Supported by PO1HL51971.
Abstract 255: Allelic Variants in Arhgef11 via the Rho-Rock Pathway Promote Epithelial-Mesenchymal Transition in Proximal Tubule Cells and Contributes to Kidney Injury
Authors: Jia Zhen , Johnson Ashley C , Guo Zibiao , Garrett Michael R
Published: March 2022

The Dahl salt-sensitive (S) rat is a widely studied model of hypertension that develops progressive kidney injury characterized by proteinuria, glomerulosclerosis and tubulointerstitial fibrosis, culminating in a significant decline in kidney function. Through positional cloning approaches, Arhgef11, a Rho guanine nucleotide exchange factor for the Rho-ROCK pathway was identified as a strong candidate gene. The most likely pathophysiological mechanism (based on animal studies) relates to the development of tubulointerstitial fibrosis, suggesting that chronic activation of Rho-ROCK in tubular cells may contribute to kidney injury observed in the S rat. Arhgef11 knockdown cell lines (up to 60% knockdown) as well as primary cultures of proximal tubule cells (PTC) were studied. Knockdown of Arhgef11 resulted in reduced RhoA activity (30% decrease, 0.57 ± 0.05 vs 0.82 ± 0.06 ng/mg protein), decreased activation of Rho-ROCK pathway (40-60%) and less stress fiber formation (2-fold reduction, 2.28 ± 0.18 vs 4.39 ± 0.64 in length/width ratio) versus control. S rat PTCs exhibited increased expression of Arhgef11, activation of Rho-ROCK, and decreased uptake of FITC-albumin (5.85 ± 0.71 vs 8.25 ± 0.64 mg/g protein) compared to control (minimal congenic). Both Arhgef11 knockdown cell-lines and PTCs stimulated with the profibrotic cytokine TGFβ1 were more prone (versus control) to epithelial-mesenchymal transition as demonstrated by increased expression of N-Cadherin, Vimentin, Col1a1 and spindle shape morphology. While dysregulation of the Rho-ROCK pathway in tubular cells appear to play an important role in EMT and promote fibrosis, chronic stimulation in other kidney cell types including vascular smooth muscle cells (VSMC) and glomerular epithelial cell (GEC) could also contribute to S rat kidney injury. Thus, we are currently investigating Arhgef11 knockdown in VSMC and GEC cell-lines as well as primary cells derived from the S rat. In summary, these studies suggest that the functional significance of genetic variants in Arhgef11 may be of major importance in promoting changes in cell morphology (Rho-Rock and/or EMT), function of tubule cells, and/or act in concert with dysregulation in VMSC and GEC to promote kidney injury.
Abstract 26: PVN GaI2 Subunit Proteins - The Key to a Salt-resistant Phenotype?
Authors: Wainford Richard D , Kuwabara Jill T
Published: March 2022

Aim:
Endogenous up-regulation of PVN Gαi
2
proteins facilitates sympathoinhibition and normotension during chronic Na
+
challenge in Sprague-Dawley rats via a renal-nerve dependent mechanism. Therefore, we examined the role of PVN Gαi
2
proteins in MAP regulation during high salt-intake in intact and renal denervated (RDNX) Dahl salt-resistant (DSR) & salt-sensitive (DSS) rats and naive Brown Norway (BN) and DSSBN8 congenic rats.

Methods:
Sham (S) or RDNX DSR & DSS rats receiving a continuous i.c.v. infusion of a scrambled (SCR) or Gαi
2
oligodeoxynucleotide (25 μg/day), and naïve BN and DSSBN8 rats, were fed a 21-day normal 0.4% (NS) or high 8% NaCl (HS) diet. MAP was recorded continuously by radiotelemetry and Na
+
homeostasis was assessed via metabolic balance studies. On day-21 plasma norepinephrine (NE), autonomic function and PVN Gαi
2
protein levels were determined (N=6/gp).

Results:
HS-intake did not alter MAP, suppressed plasma NE (P<0.05), and evoked a site-specific increase in PVN Gαi
2
protein levels in BN & DSR (4.8 and 4.5-fold respectively, P<0.05), but not DSS rats. In DSR rats Gαi
2
down-regulation evoked rapid renal nerve-dependent hypertension (MAP [mmHg] Day 4 HS S 121±2* vs RDNX 101±2, Day 21 HS S 127±2* vs RDNX 101±1), Na
+
retention and sympathoexcitation (plasma NE [nmol/L] S 87±7* vs RDNX 37±3, peak ΔMAP to chlorisondamine [mmHg] S -68±4* vs RDNX -37±3). In DSS rats, Gαi
2
down-regulation exacerbated salt-induced hypertension (MAP [mmHg] Day 4 HS S 148±5* vs RDNX 133±4, Day 21 HS S 175±4* vs RDNX 145±3), Na
+
retention, and sympathoexcitation (plasma NE [nmol/L] S 118±9* vs RDNX 88±6) in a renal nerve dependent manner. DSSBN8 rats exhibited PVN specific Gαi
2
protein up-regulation, attenuated hypertension (MAP [mmHg] Day 21 DSSBN8 HS 142±2
τ
, DSS HS 155±4), Na
+
retention and global sympathoexcitation (plasma NE [nmol/L] DSSBN8 HS 52±4
τ
, DSS HS 87±6). *p<0.05 vs RDNX + HS,
τ
p<0.05 vs DSS.

Conclusion:
PVN Gαi
2
protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal-nerve dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Owing to SNP’s in the human GNAI2 gene (which encodes Gαi
2
) correlating with hypertension our findings have translational implications.
Abstract 27: Deficiency of Angiotensinogen in Liver Markedly Reduces Blood Pressure in Lean and Obese Male C57bl6 Mice
Authors: Yiannikouris Frederique B , English Vickie , Su Wen , Gong Ming , Cassis Lisa
Published: March 2022

Objectives:
We demonstrated that adipose tissue is a major source of elevated plasma angiotensin II (AngII) in obese hypertensive mice. The liver is the primary source of systemic angiotensinogen (AGT), the AngII precursor. The purpose of this study was to define the contribution of liver-derived AGT on obesity-hypertension in male C57BL/6 mice.

Methods and results:
Male

Agt
fl/fl

mice expressing albumin-driven Cre recombinase were bred to female mice to generate littermate control (

Agt
fl/fl

) or liver-AGT deficient mice (

Agt
Alb

). Mice (n=10-13/group) were fed a low fat (LF, 10% kcal as fat) or HF diet (HF, 60 % kcal as fat) for 16 weeks. At baseline, there were no differences in body weight or systolic blood pressure (SBP) between genotypes. Liver AGT deficiency had no effect on the development of obesity (

Agt
fl/fl

, 48.0±0.4;

Agt
Alb

, 47.5±0.4 g; P>0.05). HF-fed control mice had increased plasma AGT concentrations compared to LF-fed controls (LF:

Agt
fl/fl

, 2.0 ± 0.3; HF,

Agt
fl/fl

, 3.2 ± 0.3 μg/ml; P< 0.05). Plasma AGT concentrations were markedly reduced in liver AGT-deficient mice fed either diet (LF:

Agt
Alb

, 0.5 ± 0.1; HF:

Agt
Alb

, 0.5 ± 0.1μg/ml; P> 0.05). At 9 weeks, there was no effect of HF feeding on SBP in control mice; however, SBP of

Agt
Alb

mice was significantly decreased compared to controls fed either diet (LF:

Agt
fl/fl

, 105 ± 2;

Agt
Alb

, 90 ± 2; HF:

Agt
fl/fl

, 106 ± 2;

Agt
Alb

, 87 ± 6 mmHg, P
genotype
< 0.05, P
diet
>0.05)
.
At 15 weeks, we implanted carotid artery catheters and radiotelemetry implants into anesthetized mice. HF-fed

Agt
fl/fl

mice exhibited a significant increase in SBP compared to LF-fed controls (LF:

Agt
fl/fl

, 119 ± 4; HF:

Agt
fl/fl

, 139 ± 3 mmHg, P< 0.05). Moreover, SBP was significantly decreased in HF-fed

Agt
Alb

mice compared to HF-fed

Agt
fl/fl

controls (HF:

Agt
fl/fl

, 139 ± 3; HF:

Agt
Alb

, 85 ± 4 mmHg, P< 0.05) Surprisingly, LF-fed

Agt
Alb

mice did not survive surgery for radiotelemetry, potentially resulting from marked reductions in blood pressure.

Conclusions:
Liver AGT deficiency markedly decreases systemic AGT concentrations and blood pressure in male mice fed a LF or HF diet.
Abstract 275: Tissue Transglutaminase Stabilizes Placental AT1 Receptor and Contributes to Pathophysiology in Preeclampsia
Authors: Liu Chen , Wang Wei , Parchim Nicholas , Irani Roxanna , Ning Chen , Sun Kaiqi , Zhao Shushan , Zhao Cheng , Song Anren , Zhang Yujin , Blackwell Sean , Sibai Baha , Jin Jianping , Kellems Rodney , Xia Yang
Published: March 2022

Preeclampsia (PE) is a serious hypertensive disease of pregnancy associated with the presence of the major angiotensin receptor AT1-agnostic autoantibodies (AT1-AAs) that cause the defining PE features when introduced into pregnant mice. To understand AT1-AA-mediated receptor activation in PE, we explored the role of tissue transglutaminases (TG2), the most ubiquitous member of a group of enzymes modifying proteins by catalyzing the formation of ε-(γ-glutamyl)-lysine isopeptide bonds. TG2, a versatile protein that can also function as a G protein, is enriched in the syncytiotrophoblasts of placenta, the key organ in PE pathogenesis. Western blot analysis and IHC-IF double staining initially revealed that TG2 and transglutaminase (TG)-mediated isopeptide modification levels are increased approximately 2X in PE vs normotensive (NT) placental tissue (n=4 or 5, p<0.05). Next, we identified a similar increase in PE plasma TG activity (NT=3.1±0.5, mild PE=5.4±0.6, severe PE=9.1±0.7milliunit/ml, n=26-29, p<0.01), and the increased plasma TG activity is positively correlated with the key disease features including blood pressure and proteinuria (r
2
>0.5, p<0.01). We also found a higher level of AT1 receptor with isopeptide modification in PE placentas by immunoprecipitation and IF colocalization. A pathogenic role for TG2 in PE is suggested by
in vivo
experiments in which TG inhibitor cystamine, or siRNA-mediated TG2 knockdown, significantly attenuated AT1-AA-induced PE features in pregnant mice including hypertension (PE=160±6 vs PE+cystamine=133±3mmHg, p<0.01, n=6; PE+control siRNA=161±3 vs PE+
TG2
siRNA=137±7mmHg, p<0.05, n=5-6), proteinuria (PE=133±21 vs PE+cystamine=58±12ng albumin/mg creatine, p<0.01, n=6; PE+control siRNA=102±51 vs PE+
TG2
siRNA=26±6ng albumin/mg creatine, p<0.05, n=4), and placental isopeptide modification. In trophoblasts, AT1-AA stimulation enhanced the interaction between AT1 receptor and TG2, and resulted in increased AT1 receptor stabilization via isopeptide modification, which is further confirmed by mutagenesis studies. Taken together, our results not only identify a pathogenic role of TG2 in PE, but also shed light on a previously unrecognized mechanism of TG2-mediated GPCR stabilization.
Abstract 280: Sflt-1 is Regulated via Both Transcriptional and Post-transcriptional Modification in Preeclampsia
Authors: Wang Wei , Parchim Nicholas , Iriyama Takayuki , Luo Renna , Zhao Chen , Zhang Yujin , Blackwell Sean , Sabai Bahaeddine , Tao Lijian , Kellems Rodney , Xia Yang
Published: March 2022

Preeclampsia (PE) is a life-threatening hypertensive renal disorder of pregnancy. Soluble fms-like tyrosine kinase 1 (sFlt-1), an anti-angiogenic factor, is elevated and contributes to PE. However, molecular basis underlying its up-regulation in PE remains unclear. Here we report that hypoxia inducible factor-1α (HIF-1α), an important transcriptional factor, and U2AF65, a key molecule involved in alternative splicing, are significantly increased in the placentasof PE patients compared to the controls. Using two animal models of PE including angiotensin receptor type 1 receptor agonistic autoantibody (AT1-AA) and LIGH (a TNF-α superfamily member)-induced PE mouse model coupled with
in vivo
nanoliposome-delivery system to specifically knockdown HIF-1α and U2AF65, we found that knockdown of HIF-1α or U2AF65 significantly attenuated AT1-AA-induced hypertension (HIF-1α siRNA: 137±4mmHg; U2AF65 siRNA: 140±5mmHg, vs. AT1-AA group 164±3mmHg, p<0.05), proteinuria (HIF-1α siRNA: 41.6±3.2μg/mg, 34.9±5.3μg/mg, vs. AT1-AA group 85.9±4.8μg albumin/mg creatinine, p<0.05) and circulating sFlt-1 level. Similarly, we found that HIF-1α and U2AF65 siRNA knockdown significantly reduced sFlt-1 levels, hypertension and proteinuria in LIGHT-infused pregnant mice. Finally, using splice-specific PCR assay, we found that knockdown HIF-1α significantly reduced total Flt-1 mRNA level in the placentas of both AT1-AA and LIGHT-infused pregnant mice but no effect on the ratio of sFlt-1/ Flt-1. In contrast, U2AF65 knockdown significantly reduced the ratio of sFlt-1/ Flt-1 without an effect on total Flt-1 mRNA level in these two PE mouse models. Similar to mouse, using human villous explant cultures (HVEx), we found that HIF-1α knockdown directly reduced total Flt-1 mRNA but not the ratio of sFlt-1/ Flt-1in AT1-AA or LIGHT-treated HVEx . While, U2AF65 siRNA knock down significantly reduced the ratio of sFlt-1/ Flt-1 but no effect on total Flt-1 mRNA levels in AT1-AA or LIGHT-treated HVEx. Overall, using both human and mouse studies, we have revealed two molecular basis underlying sFlt-1 inductions in PE: 1) elevated HIF-1α directly increases sFlt-1 levels under transcriptional levels; 2) elevated U2AF65 enhances alternative splicing to increase sFlt-1 levels.
Abstract 285: Renal Inflammation and Injury Markers Correlate Directly With Cortical and Fractional Hypoxia Measured With Blood Oxygen Level Dependent (BOLD) MR in Human Atherosclerotic Renal Artery Stenosis
Authors: Sad Ahmed , Herrmann Sandra , Eirin Alfonso , Tang Hui , Crane John , Glockner James , Mckusick Michael , Lerman Lilach , Textor Stephen
Published: March 2022

Background:
Atherosclerotic renal artery stenosis (ARAS) reduces renal blood flow (RBF) and amplifies regional kidney hypoxia, but their relationship to tissue inflammation and injury markers is poorly understood. We tested the hypothesis that renal injury, reflected by renal vein levels of neutrophil gelatinase associated lipocalin (NGAL) and inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) would be magnified with severe occlusive vascular disease in humans with ARAS.

Methods:
Inpatient studies performed in patients with ARAS (n=40, 71.3±11% occlusion by CT angiography) or essential hypertension (EH) (n=30), during fixed Na+ intake and ACE/ARB Rx. Single-kidney cortical and medullary perfusion and RBF were measured using multidetector CT, and glomerular filtration rate (GFR) by iothalamate clearance. Tissue deoxyhemoglobin levels (R2*) and fractional kidney hypoxia (% of axial area with R2*>30/s) were measured by BOLD-MRI at 3T.

Results:
Single kidney RBF, cortical and medullary perfusion were reduced in the post-stenotic kidney, as was GFR. Renal vein NGAL, MCP-1 and fractional hypoxia were higher in patients with ARAS than EH. NGAL and MCP-1 levels correlated directly with cortical R2* and fractional hypoxia (r = 0.52, P <0 .0001 and r = 0.4, P = 0.0006) and with the degree of artey stenosis (r = 0.37, P = 0.04 and r = 0.35, P = 0.05). GFR correlated inversely with NGAL (r= - 0.5, P <0.0001).

Conclusions:
Our results demonstrate that elevated renal venous markers of kidney inflammation (MCP-1) and injury (NGAL) in ARAS are correlated with the severity of tissue hypoxia as measured by BOLD MR, suggesting a possible link between kidney inflammation and hypoxia.
Abstract 3: Vascular Smooth Muscle ADAM17 is Critical for End-organ Damage but not Hypertension Induced by Angiotensin II Infusion in Mice
Authors: Taro Yoshinori , Takayanagi Takehiko , Elliott Katherine J , Kobayashi Tomonori , Fukuda Yamato , Eguchi Satoru
Published: March 2022

A metalloprotease, ADAM17, mediates EGF receptor (EGFR) activation in vascular smooth muscle cells (VSMC) by angiotensin II (Ang II) leading to hypertrophy. dnADAM17 prevents vascular neointimal hyperplasia. To test if vascular ADAM17 silencing has any therapeutic potential against hypertension, we evaluated Ang II-induced end-organ damage as well as hypertension in ADAM17 flox/flox mice bred with sm22α Cre mice (Cre+/-). Upon Ang II infusion (1 μg/kg/min) for 2 weeks, control Cre-/- mice showed phenotypes of cardiac hypertrophy; HW/BW ratio (mg/g: 12.1±1.6 vs 6.2±0.4 p<0.05 n=8), cardiac echo (LVPWd mm: 0.96±0.05 vs 0.75±0.04, p<0.05) and plasma BNP (pg/mL: 1.94±0.36 vs 0.07±0.2, p<0.05) compared with saline infusion. Histological assessments demonstrated medial hypertrophy and perivascular fibrosis of coronary arteries with Ang II infusion. In contrast, these phenotypic changes were attenuated in VSMC ADAM17 silenced Cre+/- hearts with Ang II infusion; HW/BW ratio (7.0±0.8 vs 6.6±0.7 n=5), cardiac echo (LVPWd: 0.71±0.09 vs 0.65±0.09) and BNP (0.52±0.19 vs 0.03±0.003, p<0.05) compared with saline infusion. Control Cre-/- with Ang II infusion also demonstrated renal dysfunction assessed by BUN (mg/dL: 40.1±2.9 vs 23.0±2.8 p<0.05 n=6) compared with saline infusion, which was partially prevented in Cre+/- mice (30.4±2.5 vs 21.3±4.0 p<0.05). Renal fibrosis observed in Cre-/- with Ang II infusion was also attenuated in Cre+/- with Ang II infusion. However, Ang II induced hypertension in both Cre-/- and +/- mice assessed by radiotelemetry (MAP mmHg: 160±6 vs 155±7). Ang II infusion in Cre-/- enhanced ADAM17 and phospho-Tyr1068 EGFR staining in vasculatures of heart and kidney, whereas Ang II infused Cre+/- had diminished phospho-Tyr1068 EGFR staining and no ADAM17 staining in the vasculatures. qPCR analyses of cardiac ADAM17 mRNA in hearts further support these findings. In addition, IHC analyses revealed less oxidative stress and less ER stress in heart and kidney of Ang II-infused Cre-/- compared with Ang II-infused Cre+/-. These data suggest that vascular ADAM17/EGFR axis is critical for end organ damage via induction of ER/oxidative stress independent from blood pressure regulation.
Abstract 30: Nuclear Factor of Activated T Cells-5(NFAT5) Plays a Protective Role in Renal Ischemia Reperfusion Injury(IRI)
Authors: Hao Shoujin , Bellner Lars , Darzynkiewicz Zbigniew , Ferreri Nicholas R
Published: March 2022

Recovery from acute kidney injury includes restoration of GFR and tubular repair but may be associated with impaired renal hemodynamic and natriuretic responses that contribute to the development of salt-sensitive hypertension and chronic kidney disease. NFAT5, a transcription factor that protects the kidney from hypertonic stress also is activated by hypoxia, however, a functional role for this transcription factor in IRI has not been established. Mice were subjected to IRI by clamping of the left renal pedicle for 30 min followed by reperfusion for 3 or 48 hr. After 3 hr, analysis by qRT-PCR showed that the relative abundance of NFAT5 mRNA was similar in mTAL tubules, or outer medulla (OM), prepared from contralateral and clamped kidneys. However, after 48 hr NFAT5 mRNA accumulation increased approximately three-fold in both mTAL tubules (NFAT5/β-actin, 3.14 ± 0.32 to 0.95 ± 0.11, p<0.05) and OM (2.71 ± 0.43 to 0.82 ± 0.22, p<0.05). Mice were then either pretreated for 72 hr with an intrarenal injection of a lentivirus shRNA construct to knockdown functional NFAT5 (EGFP-U6-N5-ex8, n=6), or a control vector (EGFP-U6, n=6) before induction of IRI. NGAL and Kim1, established biomarkers of IRI, mRNA was dramatically increased after IRI and was further increased after knockdown of NFAT5 (NGAL: EGFP-U6-N5-ex8; 111.5 ± 10.9, EGFP-U6; 59 ± 6.2, p<0.05 and Kim1: EGFP-U6-N5-ex8; 25.8 ± 3.8, EGFP-U6; 12.9 ± 2.1, p<0.05), suggesting that inhibition of NFAT5 exacerbates damage in the TAL after IRI. Apoptotic cells in OM of the clamped kidney, with or without inhibition of NFAT5, were detected by TUNEL assay and quantitated by laser scanning cytometry. The number of apoptotic cells in the OM increased nearly 2-fold after knockdown of NFAT5 (%TUNEL to PI: EGFP-U6-N5-ex8; 34.5 ± 6.3, EGFP-U6; 19.7 ± 2.4, p<0.05). These data are consistent with H&E staining showing exacerbation of histological features including loss of brush border, flattened tubular epithelium, tubular dilation, tubular lumen debris, and interstitial inflammatory cell infiltration after knockdown of NFAT5. Collectively, the data suggest that NFAT5 is part of a protective mechanism in the TAL and OM that limits renal damage induced by IRI, and may attenuate long-term detrimental renal effects associated with IRI.
Abstract 308: Genetic Interference with Peroxisome Proliferator-Activated Receptor ? (PPAR?) in Smooth Muscle Augments Cerebrovascular Effects of Angiotensin I
Authors: De Silva Michael , Modrick Lisa , Ketsawatsomkron Pimonrat , Sigmund Curt D , Faraci Frank M
Published: March 2022

Angiotensin II (Ang II) plays a major role in vascular disease, including both large and small vessel disease in brain. PPARγ is a ligand-activated nuclear receptor that exerts protective effects within the vasculature. Although studies using high affinity PPARγ agonists [eg, thiazolidinediones (TZD)] suggest interactions between PPARγ and the renin-angiotensin system (RAS) exist, the importance of these interactions in the absence of TZD treatment is less clear. We examined the hypothesis that interference with PPARγ in vascular muscle would alter vascular effects of angiotensin I (Ang I) and Ang II. Ang I has little intrinsic biological activity, being functionally important only after conversion to Ang II, a key effector of the RAS. We studied pressurized cerebral arteries (resistance vessels) in vitro from transgenic mice expressing a dominant negative mutation in human PPARγ (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). The approach allows cell-specific interference with normal PPARγ-dependent effects. Ang I (0.01-100 nM) produced only modest constriction in arteries from non-transgenic mice (eg, maximum reduction in diameter of 8±1% at 100 nM). In contrast, vasoconstriction to Ang I was increased ~4-fold in S-P467L mice (32±2% at 100 nM, P<0.01). Augmented vasoconstrictor responses to Ang I in S-P467L mice were abolished by losartan (1 μM, an inhibitor of AT1 receptors). Captopril [10 μM, an inhibitor of angiotensin converting enzyme (ACE) which converts Ang I to Ang II] completely blocked responses to low concentrations of Ang I, but only reduced effects of 100 nM Ang I by ~40%. In contrast to effects of Ang I, vasoconstrictor responses to Ang II (0.01-10 nM) and KCl (50 mM) were similar in non-transgenic and S-P467L mice (P>0.05). Vascular effects of Ang II in S-P467L mice were also abolished by losartan. These data provide the first evidence that interference with PPARγ in vascular muscle augments vascular effects of Ang I through apparent changes in both ACE-dependent and -independent metabolism of Ang I. Potent vascular effects of the precursor peptide following impairment of normal PPARγ function may exacerbate progression of Ang II-dependent vascular disease.
Abstract 313: Effects of Angiotensin II-Induced Hypertension and HFHS-Induced Obesity on PVAT Function in Young Rats
Authors: Szasz Theodora , Nunes Kenia P , Webb R. Clinton
Published: March 2022

The perivascular adipose tissue (PVAT) is a metabolically active structure that modulates the contractile, proliferative and migratory functions of underlying smooth muscle. In normal conditions, PVAT decreases contractile responses non-specifically in various vascular beds. Alterations in the secretory properties and consequently function of PVAT occur in various cardiovascular diseases, such as hypertension and metabolic syndrome. We have previously observed that contrary to genetic or long term diet-induced obesity, in a model of short-term high fat high sucrose (HFHS) diet-induced obesity, the anti-contractile effects of PVAT were increased. Animal models of hypertension, including angiotensin II-induced hypertension, are associated with a loss of anti-contractile effects of PVAT. Thus, we hypothesized that angiotensin II-induced hypertension opposes the effects of HFHS-induced obesity on PVAT anti-contractile function. We used young control non-treated rats, HFHS obese rats, angiotensin II hypertensive rats, and AngHFHS obese hypertensive rats and compared contractile function in the absence or presence of PVAT in mesenteric resistance arteries. Systolic blood pressure was significantly increased in AngII and AngHFHS rats compared to control (AngHFHS=153±17, AngII=170±15, Con=97±6 mmHg), while not significantly different from control in HFHS rats. Visceral adipose tissue mass was overall significantly increased in HFHS and AngHFHS rats compared to control rats (e.g. retroperitoneal fat AngHFHS=0.47±0.07, HFHS=0.64±0.14, Con=0.23±0.04 % body weight). In the absence of PVAT, contractile responses to PE, 5HT and ET-1 in mesenteric artery were not altered by HFHS obesity alone, however were increased in AngII and AngHFHS conditions. Anti-contractile effects of PVAT were significantly reduced in AngII hypertension but similarly increased in HFHS and AngHFHS rats (% reduction in max contraction, con: PE=13.6, 5-HT=14.04, ET-1=19.53; HFHS: PE=13.3, 5-HT=26.13, ET-1=30.93; AngHFHS: PE=36.06, 5-HT=18.20, ET-1=39.77). These data suggest that the deleterious effects of augmented BP on PVAT function are insufficient in counteracting the increase in anti-contractile PVAT effects in response to HFHS obesity in young rats.
Abstract 316: Aortic Smooth Muscle Cell Stiffness as a Mechanism for the Increased Aortic Stiffness when Aging is Superimposed on Hypertension
Authors: Sehgel Nancy L , Zhu Yi , Sun Zhe , Hunter William C , Trzeciakowski Jerome P , Vatner Dorothy E , Meininger Gerald A , Vatner Stephen F
Published: March 2022

It is generally recognized that increased aortic stiffness occurs in hypertension and aging. However, less is known about their combined influence on aortic stiffness. Furthermore, most prior studies have attributed increased aortic stiffness to changes in the extracellular matrix, whereas the hypothesis of this investigation is that a significant component of vascular stiffness resides at the level of the vascular smooth muscle cells (VSMCs), and that the contribution of this mechanism would be enhanced when aging is superimposed on hypertension. Accordingly, we examined aortic stiffness in young (16 wks) and old (64 wks) spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) wild type controls. We tested the hypothesis that the additional increase in aortic stiffness with combined aging and hypertension is mediated, in part, at the level of VSMCs. Systolic aortic pressure, measured by Millar micromanometer catheter, was significantly increased in the old compared to young SHR (191 ± 8 mmHg versus 148 ± 5 mmHg, p<0.01), but not in WKY (old, 101 ± 10 mmHg versus young, 120 ± 8 mmHg). Excised aortic ring segments were subjected to physiological levels of mechanical stretch ex vivo, and the wall stress in these ring segments was greater in older SHR than WKY (p<0.05). VSMCs were isolated from the thoracic aorta, and the stiffness of individual VSMCs was measured by atomic force microscopy nano-indentation. Isolated VSMC stiffness was 43 ± 8% greater, p<0.05, in old SHR compared to young SHR (27 ± 4 kPa). VSMC stiffness was also increased, p<0.05, in young SHR versus young WKY (14 ± 2 kPa), but this increase (92 ± 15%) was less, p<0.05, than that observed between old SHR versus old WKY (131 ± 2%). Thus, increased VSMC stiffness is a significant component of the increased aortic stiffness of hypertension, but this increase is augmented in aging hypertensive rats.
Abstract 32: Mitochondrial DNA Copy Number and Oxidative Stress in Patients with Chronic Kidney Disease
Authors: Gamboa Jorge L , Billings Frederic T , Bojanowski Matthew T , Wang Zuofei , Ikizler T. Alp , Brown Nancy J
Published: March 2022

Cardiovascular mortality increases with progressive decline in kidney function. Oxidative stress is a risk factor for cardiovascular complications in patients with chronic kidney disease (CKD). Oxidative stress also increases as renal function declines. Dysfunctional mitochondria are a main source of oxidative stress and may contribute to the pathogenesis of cardiovascular disease in patients with CKD. In this study we tested the hypothesis that mitochondrial function decreases with the progression of CKD by assessing mitochondrial DNA (mtDNA) copy number from blood mononuclear cells (PBMCs) and plasma isofurans. Isofurans formation is favored over F2-Isoprostanes when oxygen tissue tension is high, as observed in mitochondrial dysfunction. Eighty three subjects were separated in 3 groups based on eGFR: CKD stage 1-2, CKD stage 3-4, and CKD stage 5 (hemodialysis). Groups were comparable in age, gender, BMI and prevalence of hypertension. Estimated marginal mean (after controlling for BMI) of mtDNA copy number in patients with CKD stage 5 was 1.72 (95% CI 0.10-3.34), lower than in patients with CKD stage 3-4 (3.44, 95% CI 0.98-5.90, p=0.02) and CKD stage 1-2 (6.56, 95% CI 4.57-8.54, p=0.008). F2-Isoprostanes were not different among CKD groups, whereas isofurans were higher in patients with CKD stage 5 (median 59.72; IQR 43.13-91.03) compared to CKD stages 3-4 (median 48.41; IQR 30.93-66.19, p=0.01), and CKD stage 1-2 (median 39.00; IQR 26.22-67.03, p=0.005). These results suggest that progressive kidney disease is associated with mitochondrial dysfunction. Further studies are required to evaluate
in vivo
mitochondrial function in patients with different CKD stages.