bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P339: Endothelium-specific Deletion of Amyloid Precursor Protein Causes Endothelial Dysfunction of Cerebral Arteries
Authors: d’Uscio Livius V , Katusic Zvonimir S
Published: June 2021

Amyloid-β precursor protein (APP) is an integral membrane protein expressed abundantly in the endothelium of cerebral arteries. However, the exact physiological function of APP in endothelial cells is unknown. Mice with vascular endothelium-specific deletion of APP gene (eAPP-KO) were generated using loxP/Cre technology to test the hypothesis that APP plays a key role in the control of vascular endothelial nitric oxide (NO) function. Vasoreactivity of isolated basilar arteries were studied in vitro under pressurized conditions and compared to control littermates. eAPP-KO mice were normotensive (118±2 mmHg vs. control mice: 113±2 mmHg; n=5) and plasma cholesterol and blood glucose levels were not affected by endothelium-specific genetic inactivation of APP. Endothelium-dependent relaxations to acetylcholine were significantly impaired in eAPP-KO mice (maximal relaxation: 30±4% vs. 45±5% for control littermates; P<0.05; n=7-8) while endothelium-independent relaxations to NO-donor diethylamine-NONOate were unchanged (n=5-6). Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated by 31% in cerebral arteries of eAPP-KO mice (P<0.05 vs. littermates; n=6). Furthermore, basal levels of cyclic guanosine monophosphate were also significantly reduced in cerebral arteries of eAPP-KO mice (0.64±0.09 pmol/mg; P<0.05 vs. littermates: 0.97±0.11 pmol/mg; n=11). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate were not affected by genetic inactivation of APP in endothelial cells (n=5-7). Moreover, superoxide anion levels as determined by HPLC analysis of 2-hydroxyethidium were unaltered in eAPP-KO mice cerebral arteries (1.4±0.2 nmol/mg vs. littermates: 1.2±0.1 nmol/mg; P=n.s.; n=7). Our results demonstrate that impaired endothelium-dependent relaxations to acetylcholine in eAPP-KO mice are caused by the reduced expression and function of eNOS. These findings indicate that under physiological conditions APP expression in cerebral vascular endothelium plays an important role in control of endothelial function.
Abstract P340: Molecular Mechanisms of Fetuin-a-induced Vascular Dysfunction and Inflammation: Role of Oxidative Stress and Toll-like Receptor 4
Authors: Montezano Augusto C , Graham Delyth , Touyz Rhian M
Published: June 2021

Fetuin-A (FetA) is an endogenous agonist to toll-like receptor 4 (TLR4) and regulates insulin resistance and inflammation. FetA has been associated with endothelial dysfunction during metabolic diseases. Exact mechanisms whereby FetA influences vascular function in pathological conditions remain unknown, but we demonstrated that FetA regulates vascular function by Nox1 and TLR4 activation. Here we hypothesized that FetA, through changes in cell metabolism and activation of TLR4-Nox1 axis induces ROS formation and inflammation in hypertension. Normotensive (WKY) and hypertensive (SHRSP) vascular cells, as well as human microvascular endothelial cells, were stimulated with FetA (50 ng/mL). ROS production was measured by lucigenin and Amplex red, while gene expression was assessed by qPCR. FetA increased ROS production (131±49.2%), decreased H
2
O
2
intracellular levels (63±14%) and increased gene levels of IL6 (2 fold), IL1β (1 fold), RANTES (1 fold) and MMP2/9 (2 fold) in rat endothelial cells (
vs.
veh, p<0.05); all effects were blocked by TLR4 inhibitor (CLI095) and Nox1 inhibitor (ML171). FetA increased JNK (184±19%), but not p38 MAPK, activation in endothelial cells. In VSMCs, FetA-induced TLR4-dependent ROS generation was similar in WKY (136±9%) and SHRSP (144±14%) (p<0.05 vs veh). However, while IL6 gene expression was increased by FetA in WKY (4 fold) and SHRSP (0.5 fold), IL-1β gene levels were only increased by FetA in SHRSP (1 fold) derived VSMCs (p<0.05). CLI095 inhibited FetA effects on IL6 expression; however, TLR4 inhibition did not block FetA effects on IL-1β gene levels. In human endothelial cells, FetA increased ROS levels, was inhibited by CLI095 and a glucose-6-phosphate dehydrogenase (G6PD) inhibitor (6-aminonicotinamide), suggesting that FetA effects may be related to control of cell metabolism. In conclusion, FetA seems to regulate ROS and pro-inflammatory responses by TLR4, Nox1 and G6PD in endothelial cells. In VSMCs, FetA effects on oxidative stress and markers of cell injury are partially dependent on TLR4 activation and may involve other molecular partners.
Abstract P343: Exercise-induces Mitochondrial Remodeling Prevents Angiotensin II-induced High Blood Pressure
Authors: Park Joon-Young , Kim Boa , Eguchi Satoru , Rizzo Victor , Brown Michael
Published: June 2021

Objective:
Regular practice of exercise is one of the most effective non-pharmacological interventions that improve vascular health, which is thought to be mediated by a repeated exposure of vessel walls to increased hemodynamic shear stress. The objective of this study was to investigate the effect of exercise preconditioning on endothelial mitochondria in an Ang II-induced hypertension model.

Methods:
In
in vitro
experiments, human aortic endothelial cells (HAECs) pre-exposed to laminar shear stress (LSS) (20 dyne/cm
2
, 48h) or situated under static flow were incubated with Ang II (100 nM, 2-6 h). In
in vivo
experiments, mice were singly housed with or without a voluntary running wheel for 7 weeks. Ang II (1 mg/kg per day) was infused using an osmotic pump for the last 2 weeks. Saline was used as a control.

Results:
Significant increase in the expression levels of key genes related to mitochondrial biogenesis and dynamics were observed under shear stress conditions. Mitochondrial content determined by mtDNA content and immunostaining were higher (2-fold,
P
< 0.01) in the shear-exposed cells.
en face
staining showed significantly higher mitochondrial content in the conduit and muscle feed arteries from exercise-trained mice compared with sedentary controls (
N
= 10,
P
< 0.01) but not in the mesenteric arteries. Interestingly, LSS preconditioning attenuated Ang II-induced mitochondrial dysfunction in HAECs, which was evidenced by decreased mitoROS generation, increased ΔΨm, and reduced mtDNA damage. Likewise, in aortic tissues, Ang II-induced mitochondrial phenotypic changes (i.e. mitoROS production, mtDNA damage and ΔΨm reduction) were significantly reduced in exercise-preconditioned mice compared to sedentary controls. Moreover, exercise preconditioning completely blocked Ang II-induced blood pressure elevation assessed by telemetry (MAP mmHg: 160.5 ± 1.5 vs 115.0 ± 11.4).

Conclusion:
Taken together, high-magnitude LSS improves endothelial function by enhancing mtDNA integrity and mitochondrial function. These findings further support the idea that aerobic exercise is a prominent life-style modification strategy to prevent hypertension by targeting dysfunctional mitochondria in the vessel wall.
Abstract P347: Retinol-binding Protein 7 (RBP7) is Required for PPARG-mediated Endothelial Protection via Adiponectin
Authors: Hu Chunyan , Keen Henry L , Lu Ko-Ting , Davis Deborah R , Liu Xuebo , Wu Jing , Vogel Silke , Quelle Frederick W , Sigmund Curt D
Published: June 2021

PPARγ protects against endothelial dysfunction by regulation of unknown target genes. One such target, RBP7, an intracellular fatty acid-binding protein, exhibits endothelium-specific expression, but its effect on vascular function remain unknown. We hypothesize that RBP7 is endothelial protective. We examined vascular responses in basilar artery (pressurized myograph) of RBP7-knockout (KO) and wild type (WT) mice fed normal chow (ND) or high fat diet (HFD) for 8 wks. Endothelium-dependent acetylcholine (ACh)-induced relaxation was significantly impaired in HFD-fed KO mice (ACh, 100μM: 33±7% KO vs 83±10% WT, p<0.05), but not in ND-fed groups. This response was ameliorated by pre-incubation with superoxide scavenger tempol (1mM) or PEG-superoxide dismutase (100 U/ml). Mean arterial pressure (measured by radiotelemetry), body weight, hepatic steatosis, fasting glucose, glucose tolerance, and insulin sensitivity were similar in HFD-fed KO and WT mice. To identify targets downstream of RBP7, RNA-Sequencing was performed on carotid arteries from 8-week HFD-fed WT and KO mice as well as ND-fed age-matched littermates. Adiponectin (AdipoQ), a PPARγ target, was increased ~6-fold in HFD-fed WT mice, a response that was markedly blunted in KO mice. RNA sequencing was confirmed by qPCR. There was no difference in plasma AdipoQ. AdipoQ protein is expressed in endothelial cells of carotid arteries and its level of expression was increased in HFD-fed WT but not KO mice (AdipoQ/CD31: 1.14±0.1 WT-HFD vs 0.82±0.1 WT-ND, p<0.05; 0.79±0.1 KO-HFD vs 0.81±0.04 KO-ND). This led us to hypothesize that AdipoQ is involved in RBP7-mediated endothelial protection. Incubation of basilar artery with mouse full-length AdipoQ protein (5 μg/mL, 4 hours) significantly ameliorated endothelial dysfunction (ACh, 100 μM: 56±6% AdipoQ+KO vs 26±3% KO, p<0.05) and blunted carotid artery superoxide production in HFD-fed KO mice. AdipoQ also protects against endothelial dysfunction caused by subpressor Ang-II in KO mice. We conclude that RBP7 protects the endothelium from oxidative stress-induced dysfunction through an AdipoQ-dependent mechanism. Our evidence suggests RBP7 is an essential cofactor for activation of some PPARγ target genes in the endothelium.
Abstract P601: Menopausal Female Mice are Hypersensitive to Pathological Cardiac Remodeling
Authors: Konhilas John P , Pollow Dennis P , Romero-Aleshire M J , Sanchez Jessica N , Constantopoulos Eleni , Brooks Heddwen L
Published: June 2021

Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. Loss of estrogen is detrimental; yet, estrogen replacement as cardioprotective remains controversial. Gonadectomized rodents demonstrate benefit of estrogen replacement for CVD. Human studies that show benefit of hormone replacement therapy are challenged by studies that do not show benefit. A novel model of menopause utilizing 4-vinylcyclohexene diepoxide (VCD) induces gradual ovarian failure, preserving the “perimenopause” transitional period and androgen secreting capacity of residual ovarian tissue. We hypothesize that menopausal females are hypersensitive to CVD-induced pathological cardiac remodeling. To address this hypothesis, we instigated menopause in 2 month-old females by daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days); control mice received sesame oil as vehicle. Vaginal cytology was used to monitor estrous cycles and determine when cycling ceased. Mice were considered menopausal after 15 consecutive days in persistent diestrus, non-cycling. Female mice from cycling, perimenopausal and menopausal groups received the hypertensive agent angiotensin II (Ang II, 800 ng/kg/min via alzet s.q. mini-pump, 14 days). Menopause did not impact systolic blood pressure (SBP) measured via tail cuff (control: 115.1±6.0 mmHg, n=7; menopause: 115.0±5.2 mmHg, n=10). Ang II infusion induced a significant exacerbation of hypertension (SBP) in menopausal females (156.9±4.8 mmHg; n=10) compared to Ang II controls (114.7±5.0 mmHg; n=8). This hypersensitivity to Ang II-induced hypertension was attenuated by estrogen supplementation started during perimenopause (0.1 mg, 60 day release). In addition, menopausal females demonstrated worsened pathological cardiac remodeling measured by functional (echocardiography), cellular (myocardial fibrosis) and molecular (fetal gene program) assessments. Using a novel model of menopause (VCD) combined with infusion of the hypertensive agent Ang II, we demonstrated that menopausal mice are more susceptible, or hypersensitive to pathological cardiac remodeling compared to cycling and perimenopausal mice.
Abstract P602: Reduced Symptoms And Improved Heart Rate Variability Associated With Use Of Closed-Loop Noninvasive Neurotechnology By Migraineurs
Authors: Tegeler Catherine L , Shaltout Hossam A , Tegeler Charles H
Published: June 2021

Introduction:
Migraine is associated with impaired autonomic function, reduced heart rate variability (HRV), increased sympathetic activity, and symptoms of insomnia and depression. High-resolution, relational, resonance-based, electroencephalic mirroring (HIRREM) is a noninvasive, closed-loop acoustic stimulation neurotechnology that identifies dominant brain frequencies and translates them into audible tones, to support self-optimization of brain rhythms.

Objective:
We have reported use of HIRREM is associated with improved autonomic balance in a diverse cohort. We explored for effects of HIRREM in a cohort with self-reported migraine enrolled in an IRB-approved open label feasibility study of HIRREM for diverse neuropsychological disorders.

Methods:
Fifty-two subjects (42 female), mean (SD) age 38.0 (18.6), received 15.9 (3.9) HIRREM sessions (90-120 minutes each) over 9.0 (2.7) days of in-office intervention. Outcomes collected before (V1), and 13.6 (14.4) days after HIRREM completion (V2) included measures of autonomic regulation (baroreflex sensitivity, BRS, and HRV), inventories for insomnia (ISI), depression (CES-D), traumatic stress (PCL-C), and headache (MIDAS). Paired t-tests were performed. Measures of BRS and HRV (n=52) improved from V1 to V2, including HF Alpha (+8.0 ms/mmHg (SE 2.2), p<0.0012), SDNN (+6.1 ms (1.9), p=0.002), and rMSSD ms (+7.6 (2.5), p=0.004). Sympathetic tone to blood vessels and mean arterial pressure were significantly reduced. There were improvements in symptoms at V2, including ISI (n=52): -6.2 (5.7), p<0.0001; CES-D (n=38): -8.0 (9.8), p<0.0001; PCL-C (n=30): -8.2 (11.3); p<0.001; and MIDAS (n=33): -14.9 (41.7), p<0.01). No serious adverse events were reported.

Conclusions:
This exploratory study shows improved measures of autonomic balance, reduced sympathetic tone, improved sleep and mood. Data suggest that HIRREM is a promising intervention that merits further investigation to mitigate the myriad effects of migraine
Abstract P609: Saccharina Japonica Soaked In Vinegar Remarkably Decreased Blood Pressure In 2-kidney, 1-clip Renovascular Hypertensive Rats.
Authors: Maruyama Saki , Segawa Yukiko , Hashimoto Hiroko , Osera Tomoko , Kurihara Nobutaka
Published: June 2021

Objective:
One of foods indispensable to Japanese cuisine “Washoku” is algae, including Saccharina japonica (SJ) and Undaria pinnatifida. The intake of SJ is reported to decrease blood pressure (BP) in spontaneously hypertensive rats in some studies, and in 2-kidney, 1-clip hypertensive (2K1C) rats in our studies. Since SJ soaked in vinegar is often used in Japanese cuisine, we observed the effects of dietary intake of SJ soaked in vinegar on BP in 2K1C rats.

Methods:
Male Sprague-Dawley rats (6 wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, the rats started receiving a control diet (C), a diet with 5.0% (w/w) SJ (S), or a diet with 5.0% SJ soaked in 5.0% (v/v) vinegar (SV). Systolic BP (SBP) was measured by a tail-cuff method every week for 6 weeks. At the end of the protocol, mean arterial BP (MAP) was measured in each rat under anesthesia.

Results and Discussion:
Analysis of variance shows that SBP was significantly higher in 2K1C-C than SHAM-C through the experimental period (P<0.001), and that SBP was lower in 2K1C-S (P<0.05) and -SV (P<0.001) than in 2K1C-C (Fig). It also demonstrated that 2K1C-SV provided a significant reduction in SBP compared with 2K1C-S (p<0.001). At the end of the protocol, MAP in 2K1C-C was significantly higher than SHAM-C (154±4 vs 141±4 mmHg, P<0.05). Compared with 2K1C-C, a significant reduction in MAP was observed not in 2K1C-S (143±2 mmHg) but in 2K1C-SV (133±4 mmHg, p<0.05). Soaking in vinegar might bring alginate, which is one of possible components playing an important role in decreasing BP by SJ, to a low molecule and enhance the effect.

Conclusion:
SJ soaked in vinegar may decrease BP more than SJ in 2K1C rats.
Abstract P611: Immortalization of Sheep Proximal Tubule Cells Retain the Ability to Internalize Angiotensinogen that Traffics to the Mitochondria and Nucleus
Authors: Cruz-Diaz Nildris , Su Yixin , Rose James C , Wilson Bryan A , Chappell Mark C
Published: June 2021

Although there is compelling evidence for an intracellular renin-angiotensin system (RAS) that includes localization of AT1, AT2 and AT7/Mas receptors (R) on the nucleus and mitochondria of various cell types, the mechanism for the intracellular expression of angiotensins remains equivocal as the precursor protein angiotensinogen (Aogen) enters the secretory pathway upon synthesis. Proximal tubules (PTs) of the kidney present a unique cell system since the PTs internalize Aogen and transgenic mice lacking either the PT protein transporter megalin or liver Aogen exhibit reduced renal content of both Aogen and Ang II. We reported that isolated sheep PTs readily internalize Aogen, and subcellular fractionation revealed that Aogen was evident in the nuclear and mitochondrial fractions. The present study sought to establish a permanent cell line derived from the sheep PT to facilitate the characterization of Aogen internalization and processing. Sheep PT cells were isolated by protease digestion and Percoll density gradient separation, maintained in culture to promote epithelial cell growth and immortalized by SV-40 transfection. A clone (SPT-1) was obtained that expressed the SGLT-2 protein, a selective PT marker. SPT-1 cells were incubated with recombinant
125
I-Aogen at 37°C in DMEM/F12 media. A time course [0.5 to 6 hrs] revealed linear uptake of Aogen [r = 0.995] that did not saturate by 6 hrs. Pre-treatment of the SPT-1 cells with renin/ACE/neprilysin/chymase inhibitors [INHIB] or AT1R/AT2R/AT7/MasR antagonists [ANTAG] failed to attenuate Aogen internalization [Control: 209 ± 22; INHIB: 200 ± 21; ANTAG: 217 ± 15 fmol/hr/mg, n=3] while Ang II or Ang-(1-7) [10 μM, each] also did not inhibit, but tended to increase Aogen uptake [238 ± 24 and 244 ± 15 fmol/hr/mg, respectively, n=3]. Subcellular fractionation studies revealed that 12.0 ± 0.2% [n=3] of the total internalized Aogen was localized to the mitochondrial fraction with a higher content in the nucleus following an 18 hr uptake. We conclude that the established SPT-1 cell line which retains the capacity to internalize Aogen and expresses a similar pattern of protein trafficking to isolated PTs, may constitute a relevant model to elucidate the pathway for intracellular expression of angiotensins.
Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis
Authors: Satou Ryousuke , Katsurada Akemi , Miyata Kayoko , Derbenev Andrei , Zsombok Andrea
Published: June 2021

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.
Abstract P616: Evidence That The Extracellular Domain Of Na
+
/K
+
Atpase Is The Receptor For Cyclic Gmp-Induced Natriuresis
Authors: Kemp Brandon A , Gildea John J , Howell Nancy L , Keller Susanna R , Carey Robert M
Published: June 2021

Previous studies from our laboratory have shown that extracellular renal interstitial (RI) cyclic guanosine 3’5’-monophosphate (cGMP) increases urine sodium (Na
+
) excretion (U
Na
V) at the renal proximal tubule (RPT) in rats via activation of Src family kinase. Extracellular cGMP engenders this response through an unknown receptor. We hypothesized that cGMP binds to the extracellular domain of Na
+
/K
+
-ATPase (NKA) on basolateral membranes of RPT cells inhibiting Na
+
transport. In the present study, we evaluated the effect of RI infusion of rostafuroxin (RF), a digitoxigenin derivative that specifically displaces oubain (OUA) binding from NKA, on U
Na
V in the presence of RI cGMP infusion. Volume expanded, uninephrectomized, 12-week-old female Sprague-Dawley rats received RI infusions of vehicle (D
5
W) (N=8), RI cGMP (18, 36, and 72 μg/kg/min; each dose for 30 min; N=10), or RI cGMP + RF (0.012 μg/kg/min; N=5) for 90 min following a 30 min control period with RI infusion of vehicle D
5
W. RI cGMP infusion induced a significant natriuresis from 0.39 ± 0.06 μmol/min to 1.03 ± 0.21 (P<0.05), 1.17 ± 0.19 (P<0.01), and 1.94 ± 0.16 (P<0.001) μmol/min at 18, 36, and 72 μg/kg/min cGMP, respectively. RI co-infusion of cGMP + RF abolished the cGMP-induced natriuresis at all doses (F=16.05, P<0.001). There was no change in mean arterial pressure during any infusion. To further demonstrate that cGMP binds to NKA, we performed a series of competitive binding studies in isolated RPTs from normal rat kidneys (N=4 for each) with bodipy-OUA (2 μM) + cGMP (10 μM) and 8-[Biotin]-AET-cGMP (2 μM) + OUA (10 μM). In the presence of cGMP, bodipy-OUA fluorescence intensity was reduced from 1422.1 ± 63 to 1072.5 ± 64 relative fluorescent units (RFU, P<0.01). In the presence of OUA, 8-[Biotin]-AET-cGMP staining was reduced from 1916.3 ± 144 to 1492.2 ± 84 RFU (P<0.05). Serving as control, biotinylated cAMP (N=2) did not demonstrate any fluorescence above background. Together, these data suggest that cGMP may compete with RF for binding on NKA and that the extracellular domain of NKA may serve as the receptor for cGMP-induced natriuresis.
Abstract P619: A Comparison of the Adrenergic System within the Perivascular Adipose Tissue in Two-High Fat Fed Models of Obesity-induced Hypertension: Dahl S and Sprague-Dawley Rats
Authors: Ayala-Lopez Nadia , Garver Hannah , Thelen Kyan , Burnett Robert , Contreras Andres , Fink Gregory D , Watts Stephanie W
Published: June 2021

Increased sympathetic activity is one cause of obesity-induced hypertension. Over-activity of an adrenergic system in mesenteric perivascular adipose tissue (MPVAT) could contribute to high BP given its close proximity to splanchnic arteries and veins. We tested the hypothesis that high fat (HF) fed models of obesity-induced hypertension have elevated norepinephrine (NE) in MPVAT, increasing exposure of arteries to NE. Male Dahl S and Sprague-Dawley (SD) rats were fed a HF (60% fat, 0.3% NaCl; kcal) or a normal fat diet (NF; 10% fat, 0.25% NaCl; kcal) from weaning age (n=5). At 20-29 weeks of age, rats were sacrificed and tissues collected (all results shown in the table). HF increased the body weight of Dahl but not SD. Total fat mass was increased in the HF vs NF rats of both models. Mean arterial BP measured by radiotelemetry was elevated in the Dahl S HF vs NF and slightly elevated in the SD HF vs NF rats. Plasma NE was not elevated in either model. Surprisingly, MPVAT had significantly less NE in the Dahl S HF vs NF but was not altered in the SD. Expression of genes involved in NE synthesis, uptake and metabolism was measured by PCR to determine whether the MPVAT’s adrenergic system was altered in HF rats. Tyrosine hydroxylase (
Th
) mRNA was not detected in the Dahl S (SD not measured). Expression for the NE metabolizing enzymes monoamine oxidase-A (MAO-A) and catechol-o-methyl transferase (
Comt
) was not different. However, S
lc22a3
mRNA (organic cation transporter 3) was reduced in the SD HF vs NF rat. These data reveal that the elevation in BP in Dahl S and SD rats fed a HF diet may be due to a mechanism that is independent of elevated NE in PVAT.Funding: NIHP01HL70687, F31 HL12803501
Abstract P625: Improvement of Autonomic Function and C-Reactive Protein in Military Personnel with Traumatic Stress After Use Of a Closed Loop Neurotechnology
Authors: Shaltout Hossam A , Tegeler Catherine L , Tegeler Charles H
Published: June 2021

Objective:
Evaluate changes in autonomic cardiovascular control and inflammatory markers associated with use of High-resolution, relational, resonance-based, electroencephalic mirroring (HIRREM) in subjects enrolled in a pilot study for symptoms of military-related traumatic stress (MTS).

Introduction:
Symptoms associated with MTS include insomnia, depression, anxiety, activated inflammatory response and impaired autonomic control. HIRREM is a noninvasive, closed-loop acoustic stimulation technology that identifies dominant brain frequencies and translates them in real time into audible tones of variable pitch and timing, to support self-updating and self-optimization of brain activity.

Methods:
Eighteen service members or Veterans (1 female), mean (SD) age 40.9 (7.0), with MTS symptoms for 6 years (3.4), received 19.5 (1.1) HIRREM sessions over 12 days. Continuous recordings of blood pressure and heart rate, for analysis of baroreflex sensitivity (BRS) and heart rate variability (HRV), were done before and immediately after completion of the HIRREM intervention. Blood samples were also collected (n = 14) for measurement of catecholamines, cytokines, C-reactive protein and the renin angiotensin system (RAS) components. Paired t-tests were performed. After HIRREM, there was improved BRS measured as HF alpha (9.6 ms/mmHg, SE 3.1, p = 0.005), Sequence Down (7.6 ms/mmHg, 2.4, p = 0.005), Sequence Up (8.4 ms/mmHg, 3.0, p = 0.01), and Sequence All (7.6 ms/mmHg, 2.2, p = 0.002), as well as HRV; SDNN (12.0 ms, 3.5, p = 0.02), rMSSD (13.2 ms, 3.0, p < 0.001), LF power (1023.0 ms
2
, 346, p = 0.007), HF power (398.0 ms
2
, 142.0, p = 0.01), and total power (1420.8 ms
2
, 450.8, p = 0.005). C-reactive protein (36%, p = 0.057) was also reduced. There were no significant changes in the catecholamine, cytokines or RAS. There were no adverse events or dropouts.

Conclusions:
These interim results suggest improved autonomic cardiovascular regulation, across multiple measures of BRS and HRV, and reduction in CRP associated with the use of HIRREM for symptoms of MTS. Confirmation of these results in a larger cohort may provide important insights regarding both the mechanisms associated with the beneficial effects of HIRREM, and the functional disturbances underlying MTS.
Abstract P626: Stress-Induced Natriuresis and Blood Pressure Regulation in Patients with Meniere’s Disease
Authors: Lee Seung Su , Stewart Deborah L , Harshfield Gregory A
Published: June 2021

Objective:
Meniere’s disease is an idiopathic disorder characterized by strong attacks of vertigo, nausea, aural fullness, and fluctuating sensorineural hearing loss. Previous studies by our lab have suggested a possible role of stress-induced natriuresis and blood pressure regulation in Meniere’s disease. This study hypothesized patients with Meniere’s disease will excrete less sodium during stress, as compared to healthy individuals, and will have elevated and delayed blood pressure response.

Methods:
Twenty-seven patients with Meniere’s disease (51.9% male; 61.1±11.5 years; 24 Caucasians, 3 African-American) were recruited for testing. Seventy-eight percent of the patients were taking medications including anti-inflammatory, diuretics, and anti-hypertensive medications. The protocol included 10 minutes rest, 20 minutes stress (competitive video game), and 10 minutes recovery. Urine samples were collected before and after the rest and stress periods. The data was compared to our previous study which has 586 healthy adolescents (49% male; 16.4±1.12 years; 222 Caucasians, 364 African-Americans) undergoing similar stress protocol.

Results:
During stress, Meniere’s patients showed lower mean change in sodium excretion, as compared to healthy individuals (Mean deltaUNaV: 0.033±0.064 mEq/L vs 3.256±6.259 mEq/L with p=0.035, controlling for medication and age). A trend of elevated and delayed blood pressure response to stress in Meniere’s disease patients was also observed. Mean systolic blood pressure did not significantly change (p=0.798) in Meniere’s patients from stress (132.96±2.88 mmHg) to recovery (132.21±3.03 mmHg) while it changed significantly (p<0.01) from stress (115.83±0.48 mmHg) to recovery (110.02±0.41) in healthy individuals.

Conclusion:
This study, to our knowledge, is the first to consider the stress-induced natriuresis among Meniere’s disease patients. Decreased sodium excretion with elevated and delayed blood pressure response, despite taking anti-hypertensive medications, suggests that angiotensin, an important stress hormone, may play a role in the impaired natriuresis and blood pressure regulation in patients with Meniere’s disease.
Abstract P629: The Influence Of Religiosity Index And The Knowledge About Hypertension: How To Identify Indicators Of Patient With Blood Pressure Control
Authors: Oliveira Jefferson C , Bortolotto Luiz A , Vieira Margarida M , Wen Chao L , Tsunemi Miriam H , Giorgi Dante M , Hong Valéria , Fistarol Isabela R , Chiavegato Renato , Guerra Grazia M
Published: June 2021

Introduction:
Can religious beliefs associated with the prior knowledge about the disease and treatment promote therapy adherence in hypertensive patients?

Objective:
To identify association between the religiosity index (DUREL), level of education and performance on the knowledge test and blood pressure control.

Method:
Cross-sectional study of a quantitative approach, were eligible 63 hypertensive patients for which knowledge tests were used, Morisky Green, and the Religion Index (DUREL). The research was approved by the Ethics and Research Committee. The surveys were applied on the occasion of the nursing consultation and measurement of blood pressure (BP) of Office and by Ambulatory Blood Pressure Monitoring (ABPM)

Results:
Regarding the socio demographic characteristic predominated in this study: female 55.6%, the average age of 53.48±10 years, high school complete 31.7%, Catholic religion 79.4%, ethnicity 52.4% white, marital status married 66.7%, average BMI 30.14±5 kg/m
2
. In BP Office showed average systolic blood pressure (SBP) 153.58±27 mm/Hg) and the diastolic blood pressure (DBP) 91.38 ± 15 mm/Hg. The mean values of SBP obtained with ABPM was 148.93±19 mm/Hg and 91.78±15 mm/Hg to DBP at day time, and 135.78 ±18 mm/Hg to SBP and 79.33 ±15 mm/Hg to DBP sleep time. In relation to the test of Morisky-Green to correlate with the values of the BP Office showed statistical significance (p = 0.004), for patients with therapy adherence in relation to the SBP; identified 20 adherent patients (score=4) vs non-adherent patients 43 (score ≤ 3). In relation to the blood pressure values of ABPM showed statistical significance also for SBP in the sleep time (p=0.000), and DBP (p= 0,017) the Morisky test. In relation the analysis of the performance test of prior knowledge and the blood pressure values, there was no statistical significance. Concerning the association of the Religiosity index with the values of the BP Office and ABPM there was no statistical significance.

Conclusion:
The Index of Religiosity (DUREL) and the performance of prior knowledge about the disease and treatment were not sensitive to identify patients with better blood pressure control, unlike the Morisky Gren test, which was sensitive in identify patients with therapy adherence.
Abstract P630: Nlrp3 Inflammasome Activation By Mitochondrial Dna Contributes To Oxidative Stress And Inflammation In The Vasculature Of Type 1 Diabetic Mice
Authors: Pereira Camila A , Ferreira Nathanne S , Zanotto Camila Z , Carlos Daniela , Tostes Rita C
Published: June 2021

NLRP3
Inflammasome is a platform that regulates inflammatory responses by caspase-1 activation and processing of pro-IL-1β and pro-IL-18 to mature cytokines. NLRP3 is activated by several mechanisms including mitochondrial DNA (mitDNA). Circulating mitDNA is increased in diabetes, a condition associated with NLRP3 activation. We tested the hypothesis that mitDNA release is increased in type 1 diabetes (T1D) leading to NLRP3 activation and contributing to vascular inflammatory and oxidative processes. Wild type (WT) and NLRP3-deficient (NLRP3
-/-
) mice were treated with vehicle or streptozotocin (40 mg/kg),
i.p.
for 5 days. Vascular reactivity was determined in mesenteric resistance arteries (MA). Cultured vascular smooth muscle cells (VSMC) were stimulated with mitDNA of T1D (dmDNA) and control (cmDNA) mice. Caspase-1 and IL-1β activation was evaluated by western blot analysis and reactive oxygen species (ROS) by fluorescence to DHE. DNA was extracted, purified and amplified by real-time-PCR. Data are presented as mean ± standard error of mean, Veh
vs.
T1D. NLRP3
-/-
T1D mice exhibited attenuated hyperglycemia
vs.
WT T1D mice [mg/dL, 241.0±27.7
vs.
337.6±18.1, p<0.05]. MA from T1D mice exhibited decreased ACh-induced dilatation
vs.
Veh [E
max
, 46.6±4.0
vs
. 91.5±2.8, p<0.05], which was not observed in NLRP3
-/-
T1D mice. Diabetes increased vascular caspase-1 [arbitrary units (a.u.), 1.2±0.1
vs.
0.8±0.1, p<0.05)] and IL-1β activation [4.8±1.1
vs.
0.8±0.5 p <0.05], but this activation was attenuated in NLRP3
-/-
T1D. T1D mice exhibited increased NLRP3 activation and mitDNA release in pancreatic cells and increased circulating mitDNA. dmDNA, but not cmDNA, increased NLRP3 activation in VSMC (i.e. activated caspase-1 and increased IL-1β levels) [a.u., 4.2±0.1
vs.
1.9±0.1; 2.3±0.1
vs.
0.7±0.1, p<0.05]. NLRP3 activation was attenuated in NLRP3
-/-
VMSC, but not in WT VSMC incubated with a TLR-9 antagonist. Increased ROS generation was observed in response to dmDNA, which was prevented by a mitochondrial uncoupler. Our data show that T1D increases mitDNA release, which promotes vascular NLRP3 activation via mitochondrial superoxide production, contributing to T1D-associated vascular dysfunction. Financial Support: FAPESP, CNPq.
Abstract P638: Muscadine Grape Seed Extract Improves Glucose Handling in Female but not Male Hypertensive (mRen2)27 Rats
Authors: Duncan A’ja V , Tommasi Ellen N , Gallagher Patricia E , Tallant E. A , Chappell Mark C , Diz Debra I
Published: June 2021

Muscadine grapes (
Vitis rotundifolia
) are enriched in polyphenols and other flavan-3-ols that may potentially convey cardiovascular benefit through the antioxidant properties of these compounds. In the current study, we established the effects of a muscadine grape extract (MGE, Piedmont Research and Development Corp.) on blood pressure and metabolic function in 20 week-old female and male hemizygous (mRen2)27 transgenic rats, an Ang II-AT
1
R-dependent model of hypertension. Littermates were treated with MGE (0.2 mg/mL) in the drinking water for 6 weeks (n = 7; male and n=5; female); controls were given water only (n = 7; male and n = 6; female). Intraperitoneal glucose tolerance test (IPGTT) assessed glucose metabolism and serum levels of glucose and insulin were also determined. There were no significant differences between the control and MGE-treated groups for either sex in systolic blood pressure (males: 168 ± 5 vs. 179 ± 4 mmHg; females: 183 ± 5 vs. 162 ± 11 mmHg) or body weight (males: 513 ± 12 vs. 508 ± 22 g; females: 297 ± 4 vs. 294 ± 89 g). The glucose response (area under the curve - AUC) in the female MGE-treated group was markedly lower compared to the untreated controls; however, MGE elicited no effect on the glucose AUC in males (see figure). Although MGE did not influence serum insulin AUC in males or females, the MGE-treated females exhibited a trend for a lower glucose-insulin index. We conclude that MGE intake improves glucose utilization in adult female hypertensive rats independent of changes in blood pressure or body weight. The mechanism(s) underlying the differential response to MGE between the female and male (mRen2)27 transgenic remain to be established
Abstract P639: Sildenafil Treatment Improves Placental Levels of Placental Growth Factor in the Dahl Salt-Sensitive Pregnant Rat Model of Superimposed Preeclampsia
Authors: Palei Ana C , Sasser Jennifer M , Granger Joey P
Published: June 2021

Although the etiology of preeclampsia (PE) remains unclear, evidence indicates that impaired trophoblast invasion followed by placental ischemia promotes the release of placental anti-angiogenic factors into the maternal circulation. These factors then elicit maternal endothelial dysfunction and hypertension by blocking the action of molecules such as the placental growth factor (PlGF). Inhibition of phosphodiesterase (PDE)-5 with sildenafil or other has been proposed as a potential therapy for PE; however, the mechanisms whereby PDE-5 inhibitors reduce blood pressure (BP) and improve uteroplacental perfusion during pregnancy are not clear. While previous studies have shown that PDE-5 inhibition induces PlGF production from human umbilical vein endothelial cells; it is unknown whether PDE-5 inhibitors also increase PlGF from placenta. Thus, the aim of this study was to evaluate whether sildenafil enhance placental secretion/production of PlGF in vitro and in vivo. In our in vitro protocol, we incubated placental villous explants from Sprague Dawley (SD) pregnant rats (n=4, 2-3 placentas per rat) at gestational day (GD)19 with different doses of sildenafil for 48h at 37°C under normoxia (8% O
2
). PlGF-2 was measured in media of cultured explants by ELISA. We observed that sildenafil had no effect on PlGF-2 secretion from rat placental villi (vehicle: 562.7±46.6, 10nM: 559.3±39.5, 100nM: 556.4±35.9, 10uM: 546.2±37.5, and 100uM: 558.7±48.2pg/mg; P>0.05). In our in vivo protocol, we treated Dahl Salt-Sensitive (DS) pregnant rats (n=6-8 per group), which we had previously characterized as a model of superimposed PE, with sildenafil (50mg/kg per day, via food) from GD10 to 20. PlGF-2 was measured in placental homogenates by ELISA. While untreated DS dams exhibited an increase in BP and uterine artery resistance index (UARI) from baseline to late pregnancy, sildenafil-treated DS dams exhibited a significant decrease in BP and UARI. In addition, we found that placental levels of PlGF-2 were elevated in sildenafil-treated DS dams compared with untreated counterparts (1019±107.3 and 646.8±125.1pg/mg; P=0.0407). In conclusion, our findings suggest that the BP and UARI reduction in response to sildenafil may involve the indirect production of PlGF.
Abstract P641: Markers of Autophagy are Induced in Response to Placental Ischemia
Authors: Eddy Adrian C , Williams London J , Chapman Heather , George Eric M
Published: June 2021

Preeclampsia is a disorder characterized by new onset hypertension during pregnancy, as well as other clinical findings such as proteinuria and edema. This disorder affects approximately five percent of pregnancies and is the leading cause of maternal and fetal morbidity. Though the origins of the disease are unclear, it is thought that placental ischemia is central to its etiology. Commonly, the uterine spiral artery fails to adequately remodel to allow for adequate blood flow to the developing fetal/placental unit, causing hypoxia and ischemia. It is well established that hypoxia can lead to endoplasmic reticulum (ER) stress in cells. When ER stress occurs, protein misfolding occurs, leading to the unfolded protein response (UPR). One process which could be important in an attempt to restore cellular homeostasis is autophagy, a specialized process of carrying defective proteins to lysosomes for degradation to prevent aggregation from occurring. There are several factors which play a role in this process, and are utilized as a marker for autophagy, notably Beclin-1 and various autophagy related genes (Atg). Though autophagy has been examined previously in cancer and numerous cardiovascular abnormalities, it is unclear whether induction of autophagy is one of the cell survival pathways activated in response to placental ischemia. Here we have examined placental tissue from term pregnant rats and rats which have undergone placental ischemia due to the reduced uterine perfusion pressure (RUPP) procedure to to determine whether autophagy pathways have been activated as part of the cell survival response during chronic ischemia. As determined by western blot, protein levels of the autophagy markers beclin (57 ± 6 A.U. vs 113 ± 16 A.U., p<0.05), Atg3 (34 ±3 A.U. vs 54 ±8 A.U., p<0.05), Atg12 (550 ± 77 A.U. vs. 1022 ± 160 A.U., p<0.05), and Atg16 (43 ± 6 A.U. vs 147 ±47 A.U., p<0.05) were all significantly increased in placental tissue in RUPP treated animals. These data, coupled with our previous studies, suggest that several cell survival pathways are activated in response to chronic placental ischemia, notably programmed cell autophagy. The importance of these pathways in survival of placental tissue and overall placental function will be the object of future study.
Inhibiting HDAC3 (Histone Deacetylase 3) Aberration and the Resultant Nrf2 (Nuclear Factor Erythroid-Derived 2-Related Factor-2) Repression Mitigates Pulmonary Fibrosis
Authors: Chen Fang , Gao Qi , Zhang Lijun , Ding Yibing , Wang Hongwei , Cao Wangsen
Published: June 2021

Pulmonary fibrosis is a common cause of pulmonary hypertension and its development is associated with aberrant HDAC (histone deacetylase) activities and altered fibrogenic gene expressions; however, whether the epigenetic alterations causally affect pulmonary fibrosis remains poorly understood. Here, we report that HDAC3 aberration and the resultant inhibition of Nrf2 (nuclear factor erythroid-derived 2-related factor-2), a master transcription factor of antioxidative stress, contribute significantly to pulmonary fibrogenesis. HDAC3 is preferentially upregulated with concomitant Nrf2 suppression in fibrotic lungs of both idiopathic pulmonary fibrosis patients and bleomycin-treated mice. Genetic knockout of HDAC3 or Nrf2 inversely resisted or exacerbated the fibrotic pathologies, respectively, suggesting that they are crucial regulators of pulmonary fibrosis with opposite functions. Intriguingly, a HDAC3-selective inhibitor RGFP966 effectively reduced the Nrf2 suppression and normalized the fibrosis and adverse expressions of major fibrogenic proteins, Nrf2 downstream antioxidant enzymes and inflammatory cytokines. Nrf2 promoter contains a putative binding motif for FOXM1 (Forkhead box M1), a profibrotic transcriptional factor. HDAC3 and FOXM1 inducibly bound to Nrf2 promoter locus containing the motif in lung tissues of bleomycin mice, accompanied by reduced local histone3 acetylation, which were relieved by RGFP966. In cultured lung cells, bleomycin induction of Nrf2 suppression was partially attenuated by a FOXM1 inhibitor or when the FOXM1 motif was mutated; while in Nrf2 knockout mice, the antifibrotic effects of RGFP966 were largely reduced. Thus, HDAC3 aberration and its suppression of Nrf2 plays important roles in pulmonary fibrogenesis and strategies targeting HDAC3/Nrf2 axis by HDAC3 inhibition might potentially benefit patients with idiopathic pulmonary fibrosis and the related pulmonary fibrotic disorders.
Combined Therapy With Polyethylene Glycol‐20k and MCC950 Preserves Post‐Resuscitated Myocardial Function in a Rat Model of Cardiac Arrest and Cardiopulmonary Resuscitation
Authors: Liang Lian , Zhang Guozhen , Li Hui , Cheng Cheng , Jin Tao , Su Chenglei , Xiao Yan , Bradley Jennifer , Peberdy Mary A. , Ornato Joseph P. , Mangino Martin J. , Tang Wanchun
Published: June 2021

Background

To investigate the therapeutic potential of combined therapy with polyethylene glycol‐20k (PEG‐20k) and MCC950 on post‐resuscitation myocardial function in a rat model of cardiac arrest.

Methods and Results

Thirty rats were randomized into 5 groups: Sham, Control, PEG‐20k, MCC950, PEG‐20k+ MCC950. Except for sham, animals were subjected to 6 minutes of ventricular fibrillation followed by 8 minutes cardiopulmonary resuscitation. Two milliliters PEG‐20k was administered by intravenous injection coincident with the start of cardiopulmonary resuscitation; MCC950 (10 mg/kg), a highly selective NLRP3 inflammasome inhibitor, was delivered immediately after restoration of spontaneous circulation. Myocardial function, sublingual microcirculation, mitochondrial function, plasma cardiac troponin I, and interleukin‐1β, expression of proteins in SIRT1 (sirtuin 1)/PGC‐1α (peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha) and NLRP3 (the NOD‐like receptor family protein 3) inflammasome pathways were evaluated. Following cardiopulmonary resuscitation, myocardial function was compromised with a significantly decreased cardiac output, ejection fraction, and increased myocardial performance index, cardiac troponin I. Sublingual microcirculation was disturbed with impaired perfused vessel density and microvascular flow index. Cardiac arrest reduced mitochondrial routine respiration, Complex I‐linked respiration, respiratory control rates and oxidative phosphorylation coupling efficiency. PEG‐20k or MCC950 alone restored mitochondrial respiratory function, restituted sublingual microcirculation, and preserved myocardial function, whereas a combination of PEG‐20k and MCC950 further improved these aspects. PEG‐20k restored the expression of SIRT1 and PGC‐1α, and blunted activation of NLRP3 inflammasomes. MCC950 suppressed expression of cleaved‐caspase‐1/pro‐caspase‐1, ASC (apoptosis‐associated speck‐like protein), GSDMD [gasdermin d], and interleukin‐1β.

Conclusions

Combined therapy with PEG‐20k and MCC950 is superior to either therapy alone for preserving post‐resuscitated myocardial function, restituting sublingual microcirculation at restoration of spontaneous circulation at 6 hours. The responsible mechanisms involve upregulated expression of SIRT1/PGC1‐α in tandem with inhibition of NLRP3 inflammasomes.