bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P202: Impaired Central, Renal, and Blood Pressure Responses to Alterations in Fluid and Electrolyte Homeostasis in Aged Sprague-Dawley Rats
Authors: Frame Alissa A , Rodrigues Kyle R , Whelan Lillian M , Wainford Richard D
Published: June 2021

Aim:
Hypertension is strongly correlated with increased age in human subjects. These studies tested the hypothesis that impaired natriuretic responses to acute and chronic challenges to fluid and electrolyte homeostasis contribute to age-dependent hypertension.

Methods:
Two-month old (young) and 6-month old (aged) male Sprague-Dawley rats underwent an acute IV volume expansion (VE; 5% BW) and MAP, HR, urine output, and PVN neuronal activation (c-Fos expression) were assessed (n=4 per group). In a separate study, naïve 2 and 6-month old male Sprague-Dawley rats were maintained on a normal (0.6% NaCl) or high salt (4% NaCl) diet, and on day 21, MAP and NCC activity (peak natriuresis to IV HCTZ, 2mg/kg) were assessed (n=4 per group).

Results:
Renal excretion of sodium and water after acute VE was impaired in aged rats (total % sodium load excreted; young 78±6 vs aged 60±7: total % water load excreted; young 96±7 vs aged 66±5, P<0.05). PVN neuronal activation was attenuated in response to acute VE in aged rats in all parvocellular regions excluding the lateral parvocellular subnucleus (PVN neuronal activation [c-fos positive cells]; medial parvocellular young 59±4 vs aged 42±7, P<0.05). CV parameters did not change in response to acute VE in either group. Chronic HS diet evoked an increase in MAP in aged rats but not young rats (MAP [mmHg]; young NS 124±2 vs young HS 126±3 vs aged HS 138±3, P<0.05). Chronic HS diet caused a decrease in NCC activity in young rats and, in contrast, an increase in aged rats (peak ΔUNaV to HCTZ [μeq/min]; young NS 9.2±0.5 vs young HS 7.1±0.3 vs aged HS 16±1, P<0.05).

Conclusion:
Activation of sympathoinhibitory PVN parvocellular neurons is a well-characterized response to acute VE. Our data suggest there are attenuated acute PVN sympathoinhibitory responses to alterations in fluid and electrolyte balance in aged rats. In aged animals, HS intake increased NCC-mediated sodium reabsorption and promoted the development of sodium-dependent hypertension. We speculate that this is driven by blunted HS-evoked sympathoinhibition, likely due to reduced PVN neuronal activation.
Abstract P203: Renal Localization of Salt Inducible Kinase-1 and Its Regulation in Doca/salt Hypertensive Rats
Authors: Gonsalez Sabrina R , Ferrão Fernanda M , Prieto Minolfa C , Lara Lucienne S
Published: June 2021

In immortalized and primary cultured renal cells, SIK-1 is the intracellular Na
+
sensor enabling cells to enhance (Na
+
+K
+
)ATPase (NKA) activity without promoting cell swelling. In the present study, we aimed to: (1) Localize SIK-1 along the nephron; (2) To determine the effects of high salt (HS) diet and hypertension (HTN) on SIK-1 expression and activity; and (3) To examine the SIK-related impact on NKA. Adult male Wistar rats (N=60; N=15/group) underwent to unilateral nephrectomy and divided in four groups: 1) Control (CTRL); 2) CTRL/Salt (4% NaCl); 3) Deoxycorticosterone acetate (DOCA;
sc
8 mg/kg; twice a week); and 4) DOCA/Salt. After 4 weeks, systolic blood pressures (SBP) were measured by tail-cuff and then, rats were maintained in metabolic cages during 24 h for urine collections. At euthanasia, remaining kidney was removed for kidney index, histological analyses and renal cortex homogenate preparation. Regardless, salt intake, CTRL rats presented normal SBP, kidney histology, and glomerular filtration rate (GFR). However, CTRL/Salt rats exhibited augmented fractional Na
+
excretion (FENa), BUN, and proteinuria. DOCA/Salt rats presented exacerbated SBP and FENa, marked kidney injury, and decreased GFR. In all groups, SIK-1 immunofluorescence was localized in mesangial cells, thick ascending limb, and principal cells. In CTRL rats, high-salt diet increased 40% SIK-1 protein content and 4-fold SIK activity. NKA activity increased from 101±5 to 211±47 nmol Piхmg
-1
хmin
-1
in CTRL/Salt rats, but SIK inhibition brought back NKA to control values. In contrast, in DOCA/Salt rats, SIK-1 protein content did not change, SIK activity increased 2-fold, and NKA activity decreased from 77±5 to 47±6 nmol Piхmg
-1
хmin
-1
. In these rats, SIK inhibition significantly decreased NKA activity. Our data indicate that SIK-1 induces Na
+
reabsorption by increasing NKA activity but during DOCA salt-sensitive hypertension, there is SIK-1 dysregulation, which contributes in part to kidney injury.
Abstract P204: Epithelial Sodium Channel Stimulation by Glucose
Authors: Joy Parijat S , Snyder Peter M.
Published: June 2021

There is a link between diabetes mellitus and hypertension, but the underlying mechanisms are poorly understood. The epithelial Na
+
channel ENaC plays an important role in blood pressure control; ENaC mutations cause Liddle’s syndrome, an inherited form of hypertension. Previous work suggests that ENaC abundance is increased in diabetes mellitus, but the underlying mechanisms are unclear. Here we tested the effect of glucose on ENaC regulation. In Ussing chamber experiments using mouse kidney collecting duct cells (mCCD) and primary cultures of human lung epithelia, elevated glucose increased ENaC-mediated short-circuit current by 2-3 times in a dose-dependent manner from 100mg/dl to 400mg/dl of glucose. This was caused by an increase in ENaC abundance at the cell surface. We hypothesized that hyperglycemia might enhance ENaC cell surface abundance by altering activity of Nedd4-2, an E3 ubiquitin-protein ligase that binds to PY motifs within ENaC. Consistent with this hypothesis, we found that mutation of the PY motifs abolished ENaC stimulation by elevated glucose. Moreover, using a biotinylation assay, we found that elevated glucose (300 mg/dl) slowed ENaC endocytosis and reduced its degradation in the endocytic pathway. These changes in trafficking are explained by our finding that glucose reduced ENaC binding to Nedd4-2, and hence, reduced ENaC ubiquitination. O-GlcNAcylation plays a role in insulin signaling and glucose toxicity due to increased O-GlcNAcylation of target proteins. To test a role for O-GlcNAcylation in ENaC stimulation by glucose, we used 6-Diazo-5-oxo-l-norleucine (DON) to inhibit O-GlcNAcylation. DON abolished ENaC stimulation by elevated glucose. Using anti-O-GlcNAc antibody, we found that Nedd4-2 is a substrate for O-GlcNAcylation, and this modification was increased by elevated glucose. DON also reversed the reduction in binding of Nedd4-2 to ENaC at high glucose levels. Together, our data suggest a model in which hyperglycemia stimulates ENaC through O-GlcNAcylation of Nedd4-2, increasing ENaC abundance at cell surface thus increasing epithelial sodium absorption.
Abstract P205: Endothelium-specific Interference with PPARG Causes Cerebral Vascular Dysfunction in Response to Endogenous Renin-angiotensin System Activation
Authors: Nair Anand R , Mukohda Masashi , Agbor Larry N , Hu Chunyan , Wu Jing , Sigmund Curt D
Published: June 2021

Abnormal activation of the endogenous renin-angiotensin system (RAS) has been implicated in various cardiovascular (CV) disorders including hypertension, atherosclerosis and stroke. Whereas a low salt diet may be beneficial in salt-sensitive hypertension, it has been proposed to also cause CV risk due to activation of the RAS. The molecular mechanism by which RAS activation mediates vascular dysfunction remains undefined. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor which activates anti-oxidant and anti-inflammatory processes and can regulate the actions of angiotensin II (AngII) in the vasculature. We examined endothelial function in transgenic mice specifically expressing dominant-negative (DN) mutations in PPARγ in the endothelium (E-V290M) fed a low salt diet to test the hypothesis that endothelial PPARγ plays a protective role in the vasculature in response to endogenous RAS activation. Circulating levels of renin were significantly increased in both non-transgenic (NT) and E-V290M mice fed a low-salt diet for 6 weeks compared to standard chow (NT: 39.3±7.4 vs 19.8±1.3 ng/ml; E-V290M: 34.3±0.8 vs 16.0±3.8 ng/ml, p<0.05, n=5). Under baseline conditions, responses to endothelium-dependent agonist acetylcholine were not affected in E-V290M mice compared to NT (basilar artery: 66.1±11.8 vs 63.5±3.7%; carotid artery: 93.3±3.6 vs 91.1±4.2%, n=5). Six weeks of low-salt diet significantly impaired acetylcholine-mediated dilation in the basilar artery of E-V290M mice but not in NT (41.7±7.7 vs 74.2±5.0%, p<0.05, n=5). Unlike basilar artery, 6 weeks of low salt diet was not sufficient to induce vascular dysfunction in carotid artery or aorta of E-V290M mice (carotid artery: 85.6±4.4 vs 91.9±2.5%, n=5; aorta: 80.8±5.4 vs 87.0±5.6%, n=3). The responses to endothelium-independent vasodilator sodium nitroprusside (SNP) were not different in E-V290M mice compared to NT controls. We conclude that endothelial-specific interference with PPARγ causes endothelial dysfunction in response to endogenous RAS activation induced by a low-salt diet. Moreover, the cerebral circulation is particularly susceptible to low salt diet-induced dysfunction in conjunction with PPARγ impairment.
Abstract P210: (Pro)Renin Receptor Regulates Potassium Homeostasis via Intrarenal Aldosterone
Authors: Xu Chuanming , Lu Aihua , Wang Hong , Fang Hui , Zhou Li , Sun Peng , Yang Tianxin
Published: June 2021

It has been shown that transgenic overexpression of human (pro)renin receptor (PRR) results in elevated aldosterone (Aldo) level with unclear functional implications. The present study examined a potential role of renal PRR during high K
+
(HK) loading. In normal SD rats, a 1-week HK intake (5% KCl in diet) induced a 3.4-fold increase in renal protein expression of full-length PRR and 4.2-fold increase in urinary excretion of soluble PRR (sPRR). Administration of PRO20, a decoy peptide antagonist of PRR, at 700 μg/kg/d via i.p. injections, to K
+
-loaded animals elevated plasma K
+
level (5.72+0.08 vs. 4.84±0.18 mM, p<0.05) and decreased urinary K
+
excretion (2.52+0.11 vs. 3.43+0.19 mmol/24h, p<0.05), accompanied with a 26.2% reduction of urinary aldosterone (Aldo) excretion. HK downregulated NCC protein expression (57.8%) and upregulated renal protein expression of aldosterone synthase CYP11B2 (229%), ROMK (156%), calcium-activated potassium channel subunit alpha-1 (α-BK) (367%), α-Na
+
-K
+
-ATPase (596%), and β-ENaC (155%), all of which were significantly blunted by PRO20 (by 50 - 70%). The same maneuvers were applied to adrenalectomized (ADX) rats. Although plasma Aldo was extremely low and also unresponsive to HK loading, urinary Aldo excretion was elevated by 274% with this treatment, which was abolished by PRO20. The HK-induced responses of the above K
+
and Na
+
transporting proteins in ADX rats all persisted and also remained sensitive to PRO20. Additionally, spironolactone treatment in ADX rats was still effective in inhibiting kaliuresis induced by HK loading, resulting in hyperkalemia (Plasma K+: 5.13±0.07 vs. 4.19±0.27 mM, p<0.05). In primary rat IMCD cells, exposure to 10 mM KCl for 24 h augmented PRR protein expression and sPRR release in a time- and dose-dependent manner. HK upregulated Aldo release in parallel with increased CYP11B2 protein expression, which were both attenuated by PRO20 or PRR siRNA. A recombinant sPRR, sPRR-His, stimulated Aldo release and CYP11B2 expression. Taken together, we conclude that HK increased renal PRR expression that stimulates renal synthesis of Aldo that coordinates the response of renal membrane Na
+
and K
+
transporting proteins to facilitate K
+
secretion.
Abstract P211: N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) Promotes Tube Formation in Human Coronary Artery Endothelial Cells
Authors: Bordcoch Ginette , Nakagawa Pablo , Romero Cesar A , Romero Oscar A
Published: June 2021

Ac-SDKP is an endogenous peptide with anti-inflammation and anti-fibrotic effects in hypertensive and cardiovascular diseases. It is cleaved from Thymosin β4 (Tβ4) and hydrolyzed by angiotensin converting enzyme (ACE). Ac-SDKP plasma concentration increases after treatment with ACE inhibitors (ACEi) and some of the beneficial effects of ACEi treatment has been ascribed to Ac-SDKP. Ac-SDKP is a mediator of angiogenesis in
in-vitro
and
in-vivo
animal models. Ac-SDKP stimulates rodents derived immortalized aortic endothelial cells migration and capillary-like structures formation (tube formation). Similarly, Ac-SDKP increases capillary density after myocardial infarction in rats. The mechanism related to angiogenesis induced by Ac-SDKP is not known. Tβ4 (Ac-SDKP precursor) promotes endothelial cell migration and angiogenesis by the activation of the VEGF/AKT pathway. Our objective is to evaluate the Ac-SDKP pro-angiogenic effect in Human Coronary Artery Endothelial Cells (HCAEC) and the mechanism that regulates the angiogenic effect of Ac-SDKP. HCAEC do not produce VEGF, thus we hypothesize that Ac-SDKP increases VEGF expression in fibroblasts and that indirectly could promote capillary tube formation in endothelial cells. We used primary culture of rat cardiac fibroblast (RCF) and we treated these cells with 10nM Ac-SDKP for 24 hours. VEGF concentration in cell supernatant was measured by ELISA. Cells were starved without serum overnight before the Ac-SDKP treatment. For capillary tube formation assay, HCAEC cells were seeded into matrigel and incubated in presence of 10nM Ac-SDKP for 12 hours, pictures were taken by double phase contrast microscope and tube length was quantified with image J software and the results were expressed as percentage of control. After Ac-SDKP treatment, VEGF concentration did not increase in the supernatant of RCF (control: 0.12±0.07 vs. Ac-SDKP: 0.14±0.09 mg/ml; p=0.7). However, Ac-SDKP treatment induced the development of tube formation in HCAECs by 7±2% respect to control (p=0.037). We conclude that Ac-SDKP induces capillary tube formation not only in rodent but also in human derived endothelial cells. The mechanism by which Ac-SDKP promotes tube formation in HCAECs is still unknown.
Abstract P217: Mitochondrial N-Formyl Peptides Elicit Changes in Endothelial Cell Cytoskeleton via Formyl Peptide Receptor Activation
Authors: Wenceslau Camilla F , McCarthy Cameron G , Webb R.Clinton
Published: June 2021

One major pathophysiological characteristic of cardiovascular disease, including hypertension, is vascular dysfunction. Recently, we demonstrated that mitochondrial damage-associated molecular patterns are elevated in the circulation of SHR. Mitochondria carry hallmarks of their bacterial ancestry and one of these hallmarks is that this organelle still uses an N-formyl-methionyl-tRNA as an initiator of protein synthesis. We observed that mitochondrial N-formyl peptides (F-MIT) infusion into rats induces inflammation and vascular dysfunction, including vascular leakage, via formyl peptide receptor (FPR) activation. However, neutrophil depletion did not change this response. Therefore, we hypothesize that F-MIT via FPR activation elicits changes directly to cytoskeleton-regulating proteins in vascular cells, which may lead to increased vascular permeability. To test this hypothesis we used vascular smooth muscle cells (VSMC) and endothelial cells harvested from aortas of Sprague-Dawley rats (n=5) and human donors (n=5), respectively. Cells were divided into three groups for Western blot analysis of cytoskeleton-regulating proteins. The cells were incubated for 20 minutes in medium with either vehicle (non-formylated peptide), F-MIT (10 μM), or F-MIT after a 5-minute pre-incubation with FPR1 and 2 antagonists (Cyclosporine H, CsH, 1 μM and WRW4, 10 μM). In endothelial cells, the treatment with F-MIT increased the protein expression of RhoA/ROCK (Rho: 1.8 fold vs. Veh; ROCK: 1.4 fold vs. Veh, p<0.05), cell division control protein 42 (CDC42) (2.0 fold vs. Veh, p<0.05) and phospho-myosin light chain (MLC)
Thr/Ser19
(1.5 fold vs. Veh, p<0.05). These changes were all abolished in the presence of FPR antagonists. On the other hand, F-MIT decreased expression of phospho-MLC (0.6 fold vs. Veh, p<0.05) and CDC42 (0.5 fold vs. Veh, p<0.05) and did not change RhoA/ROCK expression in VSMC. In conclusion, F-MIT, via FPR activation, elicits direct changes in endothelial cell and VSMC cytoskeleton-regulating proteins. This interaction can lead to endothelial contraction, increased vascular leakage and attenuated barrier function as observed in clinical and experimental hypertension.
Abstract P218: Induction of Human Endothelin-1 Overexpression for 3 Months Causes Blood Pressure Rise and Small Artery Endothelial Dysfunction and Stiffening
Authors: Coelho Suellen C , Ouerd Sofiane , Fraulob-Aquino Julio C , Barhoumi Tlili , Offermanns Stefan , Paradis Pierre , Schiffrin Ernesto L
Published: June 2021

Background:
The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Recently, we developed a transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) using Cre/loxP technology. ieET-1 mice exhibited BP rise after three weeks of induction in an ET type A (ET
A
) receptor-dependent manner, in absence of vascular and kidney injury. It is unknown whether long-term exposure to ET-1 overexpression results in sustained BP elevation and vascular injury.

Design and methods:
Nine to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of endothelium-specific
Tie2
promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and studied 3 months later. ieET-1 mice were treated or not with ET
A
receptor blocker, atrasentan (10 mg/kg/day, PO) in the last 2 weeks of the study. BP by telemetry, endothelial function and vascular remodeling by pressurized myography and reactive oxygen species (ROS) generation using dihydroethidium staining in mesenteric artery (MA) or perivascular fat (PVAT) and renal artery flow (RAF) by ultrasonography were determined.

Results:
Systolic BP was increased in ieET-1 and normalized by atrasentan compared to ieCre mice (141±0 and 124±4 vs 120±0 mm Hg,
P
<0.001). Endothelium-dependent relaxation responses to acetylcholine were impaired in ieET-1 and uncorrected by atrasentan compared to ieCre (35±4 and 32±4 vs 65±8%,
P
<0.01). Media/lumen and media cross-sectional area were unchanged, but stiffness was increased in ieET-1 and normalized by atrasentan compared to ieCre mice (strain at 140 mm Hg: 0.6±0.0 and 0.7±0.0 vs 0.7±0.0 ΔD/D,
P
<0.05). ROS generation was enhanced in PVAT of ieET-1 and normalized with atrasentan when compared to ieCre mice (1.4±0.1 and 0.9±0.1 and vs 1.0±0.1 relative fluorescence units/μm
2
,
P
<0.05). RAF was decreased in ieET-1 and unchanged by atrasentan compared with control (1.8±0.2 and 2.0±0.2 vs 3.0±0.3 mL/min,
P
<0.01).

Conclusions:
Long-term exposure to endothelial human ET-1 overexpression caused sustained BP elevation, endothelial dysfunction and vascular stiffening and oxidative stress.
Abstract P220: Matrix Metalloproteinase 2 Knockout Protects from Angiotensin II-induced Vascular Injury
Authors: Fraulob-Aquino Julio C , Barhoumi Tlili , Mian Muhammad O , Idris-Khodja Noureddine , Huo Ku-Geng , Rehman Asia , Ebrahimian Talin , Lehoux Stéphanie , Paradis Pierre , Schiffrin Ernesto L
Published: June 2021

Objective:
Matrix metalloproteinase-2 (MMP2) participates in the mechanisms of vascular injury in atherosclerosis. Whether MMP2 plays a role in angiotensin (Ang) II-induced hypertension and vascular remodeling is unknown. We hypothesized that
Mmp2
knockout (

Mmp2
-/-

) would prevent Ang II-induced hypertension and vascular injury.

Methods and Results:
Fourteen days of Ang II infusion (1000 ng/kg/min, SC) increased systolic blood pressure (SBP, 161±9 vs 122±3 mm Hg,
P
<0.05) and decreased vasodilatory responses to acetylcholine (33±5 vs 83±3%,
P
<0.001), increased the media/lumen (4.8±0.4 vs 3.1±0.1%,
P
<0.001) and media cross-sectional area (7223±467 vs 5346±336 μm
2
,
P
<0.05) and enhanced stiffness (
P
<0.05), as shown by a leftward shift of the stress/strain relationship of mesenteric arteries in wild-type mice. Ang II enhanced aortic (73±6 vs 6±1 relative fluorescence units [RFU]/μm
2
,
P
<0.001) and perivascular adipose tissue (PVAT) reactive oxygen species generation (76±11 vs 12±1 RFU/μm
2
,
P
<0.001), aortic VCAM-1 (17±3 vs 5±1 RFU/μm
2
,
P
<0.001) and MCP-1 expression (71±14 vs 11±3 RFU/μm
2
,
P
<0.001), PVAT monocyte/macrophage (1.8±0.3 vs 0.1±0.1 % of PVAT,
P
<0.001) and T cell infiltration (56±14 vs 16±9 cells/μm
2
,
P
<0.05) and the fraction of spleen activated CD4
+
CD69
+
(17±2 vs 10±1 % of CD4+ T cells,
P
<0.001), CD8
+
CD69
+
T cells (11±1 vs 5±1 % of CD4+ T cells,
P
<0.001) and Ly-6C
hi
monocytes (53±6 vs 25±2 % event,
P
<0.001). Ang II increased phosphorylation of epidermal growth factor receptor 1.9±0.2-fold and extracellular-signal-regulated kinase 1/2 1.4±0.1-fold in vascular smooth muscle cells isolated from mesenteric arteries of wild-type mice (
P
<0.05).
Mmp2
knockout prevented or reduced all of the above (
P
<0.05) except SBP elevation. Bone marrow transplantation experiments revealed that Ang II-induced hypertension was impaired in absence of immune cell MMP2 and endothelial dysfunction was blunted or reduced in absence of immune or vascular cell MMP2 (
P
<0.05).

Conclusions:
Mmp2
knockout prevented Ang II-induced vascular injury but not hypertension. Bone marrow transplantation experiments revealed a complex relationship of immune and vascular cell MMP2 in the development of Ang II-induced hypertension and endothelial dysfunction.
Abstract P226: Endothelin-1 Overexpression Exaggerates Diabetes-induced Endothelial Dysfunction by Altering Oxidative Stress
Authors: Ouerd Sofiane , Idris-Khodja Noureddine , Mian Muhammad O , Gornitsky Jordan , Barhoumi Tlili , Paradis Pierre , Schiffrin Ernesto L
Published: June 2021

Objective:
Increased endothelin (ET)-1 expression has been shown to cause endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we sought to determine whether ET-1 contributes to diabetes-induced endothelial dysfunction. We hypothesized that overexpression of ET-1 in the endothelium will exaggerate diabetes-induced endothelial dysfunction.

Methods:
Diabetes was induced by streptozotocin IP injections (STZ, 55 mg/kg/day) for 5 days in 6-week-old male wild-type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Endothelial function and vascular remodeling using pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription-quantitative PCR were assessed in mesenteric arteries (MA).

Results:
MA endothelium-dependent vasodilatory responses to acetylcholine were reduced 24% by diabetes in WT (E
max
: 61±6 vs 84±3%,
P
<0.05), and further decreased by 12% in eET-1 (E
max
: 49±5,
P
<0.05). Diabetes decreased MA media/lumen in WT (2.4±0.1% vs 3.3±0.2%,
P
<0.05) and eET-1 (2.9±0.2% vs 4.0±0.2%,
P
<0.05), whereas ET-1 overexpression increased MA media/lumen to a similar extent in diabetic and non-diabetic WT mice (
P
<0.05). Vascular ROS production in MA was increased 2-fold by diabetes in WT (5.0±0.5 vs 2.5±0.3 relative fluorescence units [RFU]/μm
2
,
P
<0.05) and further augmented 1.7-fold in eET-1 (8.5±1.2 RFU/μm
2
,
P
<0.05). Diabetes reduced endothelial nitric oxide synthase (eNOS,
Nos3)
mRNA expression in eET-1 by 50% (0.7±0.1 vs 1.4±0.2,
P
<0.05) but not in WT. Induction of diabetes caused a 50% increase in superoxide dismutase 1 (
Sod1
, 1.5±0.2 vs 1.0±0.0,
P
<0.05) and a 30% increase in
Sod2
(1.3±0.1 vs 1.0±0.0,
P
<0.05) mRNA expression in WT but not in eET-1.

Conclusions:
Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by an increase in vascular oxidative stress, a decrease in eNOS expression and a decrease in antioxidant capacity.
Abstract P235: Balancing Truss Expression Paves a Way for Cardiac Hypertrophy Therapy
Authors: Li Hongliang , Zhang Xiao-Jing , Deng Ke-Qiong
Published: June 2021

Pathological cardiac hypertrophy, which is always accompanied by cardiac fibrosis and the resultant cardiac dysfunction, leads to hear failure and even sudden death. The TNF-receptor ubiquitous signaling and scaffolding protein (TRUSS) that is enriched in the heart has been identified as a negative regulator of cancer. However, the role of TRUSS in cardiac remodeling is unknown. Here, we aimed to investigate the potential participation of TRUSS in cardiac hypertrophy and the molecular events by which TRUSS regulates this pathological condition.

The pathological cardiac hypertrophy model was established by pressure overload
in vivo
and Ang II stimulation
in vitro
. We observed that the expression level of TRUSS was dramatically increased in the heart and in primary cardiomyocytes upon pro-hypertrophic stimuli. To illustrate the functional role of TRUSS in cardiac remodeling, the cardiac specific knockout (KO) or transgenic (TG) mice were employed. After aortic binding (AB) for 4 weeks, TRUSS deficiency conferred significant resistance to pressure overload
via
significantly inhibiting cardiomyocytes enlargement and fibrosis formation by about 37% and 46%, respectively, whereas dramatically exacerbated hypertrophy, fibrosis, and cardiac dysfunction were shown in TRUSS-TG mice compared to their littermate controls. Mechanistically, TRUSS can directly bind to JNK, a well-known pro-hypertrophic factor, and activate its downstream pathway. Further investigations indicated that the aggravated effect of TRUSS on cardiac hypertrophy can be almost completely reversed by a specific JNK inhibitor, SP600125, indicating a JNK-dependent manner of TRUSS-regulated cardiac hypertrophy. The directly exacerbated function of TRUSS in cardiomyocytes and the JNK-dependent mechanisms were further validated in primary cardiomyocytes that treated with Ang II after infection with AdshTRUSS or AdTRUSS. Notably, the increased protein and mRNA expression of TRUSS was also observed in heart samples from patients with hypertrophic cardiac myopathy. In conclusion, TRUSS functions as a positive regulator of pathological cardiac hypertrophy, suggesting a promising therapeutic approach for the hypertrophy related heart diseases by balancing TRUSS expression.
Abstract P239: Papaverine-induced Ventricular Fibrillation Developing During Coronary Flow Reserve Studies
Authors: Okabe Yoshitaka , Otowa Kannichi , Mitamura Yasuhito , Kikyoutani Manabu
Published: June 2021

Background:
Estimation of the fractional flow reserve (FFR) is considered to be an established method by which to assess stable coronary artery stenosis. Induction of maximal coronary hyperemia is important during FFR. Papaverine is often used to achieve maximal hyperemia. However, this drug has been reported to increase the risk of ventricular arrhythmias. The purpose of the present study was to discover predictors of papaverine-induced ventricular fibrillation (VF) developing during FFR.

Methods:
A total of 187 clinically stable patients were included in the study. FFR was performed to evaluate lesions for which percutaneous coronary intervention (PCI) was to be considered after coronary angiography. FFRs were determined after intracoronary papaverine administration (12 mg into the left and 8 mg into the right coronary arteries). We compared patients in whom VF did and did not develop in terms of clinical and ECG characteristics.

Results:
We performed FFR on 214 lesions (112 in the left anterior descending arteries, 38 in the left circumflex arteries, and 64 in the right coronary arteries) of 187 patients. The average patient age was 72.5 ± 10 years. We found that the QTc interval was prolonged in all patients after papaverine administration (average post-administration QTc interval = 569 ± 89 ms; average ΔQTc interval = 144 ± 80 ms). VF developed in three patients with significantly prolonged QT intervals (average post-administration QTc interval = 639 ± 19 ms, average ΔQTc interval = 220 ± 64 ms, p < 0.02) and all of them occurred after administration of papaverine into the left coronary artery. Three-vessel disease was significantly predictive of VF (p < 0.003). In the three-vessel group, the complications of low left ventricular function (ejection fraction<50%), hypokalemia (serum K <3.5 mEp/L), and bradycardia (<50 beats/min), were significantly associated with VF (p < 0.045).

Conclusions:
Three-vessel disease is a predictor of the development of VF during FFR performed with the aid of papaverine, especially if accompanied by one or more of low left ventricular function, hypokalemia, or bradycardia.
Abstract P241: Irisin Protects Mitochondria Function During Pulmonary Ischemia Reperfusion Injury
Authors: Zeng Chunyu , Chen Ken
Published: June 2021

Background:
Ischemia and reperfusion (I/R) induced lung injury is one of the most important and common causes of early and high morbidity and mortality. The mitochondrial damage of alveolar epithelium cells leads to further damage in lung I/R by augmented ROS activity and inflammation. Previous study show a humoral myokine, irisin, has been found stimulate mitochondrial biogenesis. We hypothesize irisin might protect lung from I/R injury.

Methods:
The lung I/R injury model was performed on C57BL/6J mice. The pulmonary protection of exogenous irisin was indicated by lung edema and function measurement, and the survival of mice. The localization of irisin was measured by immunohistochemistry and immunofluorescence staining. And the protective effect of irisin to mitochondrial was demonstrated by ATP production, ROS production, mitochondria-dependent apoptosis measurement, and so on.

Results:
There was no endogenous irisin expression in the alveolar wall in normal mice. However, it is interesting to find that, irisin was in the alveoli after lung I/R injury. The increased irisin in lung might be from plasma, because in the mean time, the plasma irisin levels were decreased. Exogenous intravenous injection of irisin protect the lung from I/R injury. The protective effect might be via the protection on the mitochondria in lung, exogenous irisin was found to localize in the mitochondria, the impaired mitochondrial function in I/R mice was reversed after irisin treatment. Moreover, there was colocalization between the irisin and UCP2, exogenous irisin decreased the I/R-induced UCP2 degradation. The role of UCP2 on the protection of mitochondria is further confirmed in the in
in-vivo
experiment, inhibition of UCP2 or knockout of UCP2 would make the protection of irisin on lung function and mitochondrial function lost.

Conclusions:
Administration with exogenous irisin would alleviate the I/R damage, reduced the inflammatory and superoxide factors, ameliorated the mitochondrial dysfunction, via the binding of irisin and UCP2 in lung.
Abstract P243: A New Type 2 Diabetic Rat Model That is Associated with Cognitive Impairment in Aging
Authors: Lv Wenshan , Yu Hongwei , Li Longyang , Taylor Christine , Gonzalez-Fernandez Ezekiel , Sims Jerrell , Elliott Matthew R , Wang Yangang , Williams Jan M , Roman Richard J , Fan Fan
Published: June 2021

Alzheimer’s disease (AD) is an incurable neurodegenerative disease and the most common form of dementia, and AD and type II diabetes (DM II) are two of the most common diseases of aging. Numerous studies demonstrate DM II with increased risk for dementia, however, the mechanisms linking DM II and AD have not been fully elucidated. There is increasing evidence suggesting that cerebral vascular dysfunction plays an important role in the development of AD. T2DN rat is a DM II rat model that exhibits diabetic nephropathy. The present study examines whether aged T2DN rat is associated with cognitive impairment, and whether autoregulation of cerebral blood flow (CBF) is impaired that contributes to AD. The levels of glucose (422 ± 32 vs. 94 ± 3 mg/dL) and glycated hemoglobin (HbA
1c
, 11.5 ± 0.2 vs. 4.3 ± 0.1%) were higher in 12-18 months old T2DN than in age matched SD control rats. CBF rose by 137 ± 15% and 36 ± 5%, respectively, in T2DN and SD rats when MAP was increased from 100 to 180 mmHg. Aged T2DN rats exhibited BBB leakage and “AD” like cerebral vascular remodeling. The expression of Amyloid β 42 (Aβ
4
2
), p-tau (S416), GFAP and IL-1 beta were significantly higher in the brains of T2DN vs. SD rats. T2DN rats also exhibited learning and memory dysfunction as the short term (2-hour; T2DN 96 ± 12 vs. SD 13 ± 3 seconds) and long term (24-hour; T2DN 105 ± 15 vs. SD 8 ± 2 seconds) latency of escape were longer in an eight-arm water maze test, and spent less time in the target arm 48 hours after training (T2DN 3.4 ± 2.6 vs. SD 45.0 ± 1.7%). These findings indicate that T2DN is a new type II diabetic rat model. Elderly T2DN rat is associated with an impaired autoregulation of CBF, glial activation and inflammation which may contribute to the development of cognitive impairment and AD.
Abstract P245: Contraindications and Exclusion Criteria in Guidelines for Rt-pa in Acute Ischemic Stroke: Can the New Aha/asa Guideline Expand the Use of Rt-pa?
Authors: Nathaniel Thomas I , Williams Jessica-Ashley , Fazzone Brian , Yi Sara , Morris Gabrielle , Black Leigh-Ann , Fredwall Megan , Staford Clay , Adkins Alyssa , Polk Shuler
Published: June 2021

Background:
Most of the original exclusion criteria for recombinant tissue plasminogen activator (rt-PA) from NINDS trial, European, and FDA contribute to the low rt-PA use because of their stringent criteria. The new AHA/ASA guideline suggests that by relaxing the already existing criteria the use of rt-PA could be increased from its current use. The impact of the new AHA/ASA guideline in expanding the use, outcomes and benefits of rt-PA is not clear. We determined these issues in the current study.

Methods:
We characterized absolute and relative contraindications to rt-PA using a large sample size collected between 2010 and 2013 in a stroke registry. We analyzed both imaging and rt-PA data to determine whether there would have been significant increase in use, outcomes and benefit of rt-PA if the AHA/ASA guideline was used instead of the old FDA package insert of Alterplase (Activase).

Results:
A total of 663 ischemic stroke patients were eligible to receive rt-PA. Out of the 663, 241 received rt-PA and 422 did not. We identified specific differences in outcomes and contributing factors to contraindications at the level of individual patients and stroke population. We observed clinical conditions of similar exclusion criteria but with differential stroke deficits in both AHA/ASA guideline and the old FDA package insert of Alterplase (Activase).

Conclusion:
Although the new AHA guideline may serve the purpose of helping to expand the safe and judicious use of alteplase after stroke, there are specific clinical factors that are necessary to achieve the goal of the new AHA/ASA guideline.
Abstract P254: Impact of Socially Influencing Systems on Hypertension Control
Authors: Siddiqui Khan , Goglia Ross , Ghaffar Aamer
Published: June 2021

Background:
Socially influencing systems (SIS) have shown to impact behavior change and outcomes in various clinical scenarios. Two aspects of SIS, i.e., Social Competition and Social Recognition are also known to increase engagement in a program.

Objective:
To evaluate the impact of social influence on hypertension control.

Methods:
All hypertensive patients that had initial hypertensive reading between July 1, 2015 and September 30, 2015 using an ambulatory BP kiosk (higi Station, higi SH llc) were identified. A random sample of 1,352 patients were identified as controls and 38,885 patients were invited to participate in a challenge to check their BP on a weekly basis. Weekly drawing for $25 gift card was conducted for those that checked their BP that week and a grand prize of $100 was awarded to the user with the most BP readings. Challenge duration was from October 1, 2015 to January 20, 2016. Patients who joined also received weekly email reminders to check their BP as well as coaching tips on how to maintain or improve their BP. Patients invited to join challenge but did not participate were referred to as Invitees, those who participated were referred to as Joiners and those not invited as Control.

Results:
A total of 1,655 patients participated in the challenge. Analysis of variance indicated a statistically significant difference between Control and Joiners (p=.016) as well as between Invitees and Joiners (p=0.009). Controls’ mean arterial pressure change increased 38.5% during the course of the study, while Invitees’ mean arterial pressure change increased 12.2%, and Joiners’ mean arterial pressure change dropped by almost 45%.

Conclusion:
Social competition and social recognition as implemented in the form of a BP check challenge showed significant reduction in mean arterial pressure. Incorporating socially influencing systems in treatment protocols for hypertension can assure adherence to the program and improve outcomes.
Abstract P255: Addressing Management of Resistant Hypertension: Effect of Online CME
Authors: Spyropoulos Jelena , Kearns Charles
Published: June 2021

Introduction:
Clinicians have a less than adequate understanding of how to properly evaluate patients to diagnose treatment-resistant hypertension, and how to use combination therapies and strategies for managing adherence to therapy to optimize outcomes.

Objective:
To determine if an online, video-based continuing medical education (CME) intervention could improve knowledge and competence of cardiologists and primary care physicians (PCPs) in managing patients with resistant hypertension.

Methods:
An online CME activity was developed as a 25-minute roundtable discussion with 3 leading experts on strategies to manage patients with VTE. The activity included a transcript and a downloadable slide deck to reinforce key data. The effects of education were assessed using a linked pre- /post-assessment study design. For all questions combined, the McNemar’s chi-square test was used to assess differences from pre- to post-assessment. Cohen’s d was used to calculate the effect size.

Results:
The change in correct responses from pre- to post-assessment achieved statistical significance for all 4 questions for cardiologists (N=156; d=0.981; P<.001) and PCPs (n=539; d=0.978; P<.001) with a large effect size for both specialty groups (Table).

Conclusion:
The significant improvements observed as a result of participation in this CME intervention demonstrate that well-designed internet-based education can improve knowledge and competence of physicians. However, both cardiologists and PCPs demonstrate a need for further education on the prevalence of resistant hypertension, clinical data, and strategies to address nonadherence to antihypertensive therapy.
Abstract P268: The Effects of a Slow Pressor Dose of Angiotensin II on Sodium Transporters Expression in the Kidney Cortex are Independent of PPAR-alpha
Authors: Khundmiri Syed J , Ecelbarger Carolyn M , Lee Dexter L
Published: June 2021

A slow pressor dose of Angiotensin II (Ang II) has been shown to increase the expression of sodium transporters in the proximal tubules (NHE3), TALH (NKCC2) and distal nephrons (NCC) of Sprague Dawley rats before an increase in blood pressure. Peroxisome Proliferator Activated Receptor - alpha (PPAR-alpha) has been shown to be involved in pressure natriuresis through changes in sodium transport via ameloride and thiazide-dependent mechanisms. We hypothesized that the changes in expression of the sodium transporters during Ang II hypertension were dependent upon PPAR-alpha expression. To address this hypothesis, we treated wild-type (WT) and PPAR-alpha knockout (KO) mice with a slow pressor dose of Ang II (400 ng/kg/min) for 12 days. Mean arterial pressure (MAP) was measured by radiotelemetry. Control MAP was not different between WT (110 ± 8 mmHg) and PPAR-alpha KO mice (112 ± 12 mmHg). On day 12 of Ang II, MAP for PPAR-alpha KO (156 ± 16) mice was significantly higher than WT (138 ± 11 mmHg) mice. The expression of NHE3, NHERF1, NKA-α1 subunit, NKCC2, and NCC was detected in kidney cortical homogenates by western blotting. Kidneys were homogenized and 25 μg of supernatant proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose paper, and blotted against antibodies to NHE3, NHERF1, NKA α1 subunit, NKCC2, and NCC. The slow pressor dose of Ang II decreased the expression of NHE3 in WT + Ang II (0.14 ± 0.02 ODU) and PPAR-alpha KO + Ang II (0.10 ± 0.02 ODU), when compared to WT (2.61 ± 0.93 ODU) and PPAR-alpha KO (2.20 ± 0.58 ODU) controls. Ang II-treatment also decreased NKCC2 in both WT (0.31 ± 0.10 ODU) and PPAR-alpha KO (0.22 ± 0.03 ODU). Ang II hypertension caused similar decreases in NCC and NHERF1 expression in WT and PPAR-α KO mice. NKA alpha1 subunit expression was increased during Ang II hypertension in both WT (1.06 ± 0.26 ODU) and PPAR-α KO (1.64 ± 0.26 ODU) mice. Our results suggest that the effects of a slow pressor dose of Ang II on expression of sodium transporters are independent of PPAR-alpha expression. Future studies are needed to determine the effects of decreasing NHE3, NKCC2, NCC and NHERF1 expression in the kidney on urinary salt excretion during a slow pressor dose of Ang II.
Abstract P269: Sodium Transporter Profile in Mice Lacking AT1A Receptors in the Renal Proximal Tubule
Authors: McDonough Alicia A , Izuhara Audrey K , Xiang Zhidan , Ralph Donna L , Adhikari Rishav , Gurley Susan B
Published: June 2021

We have reported that mice lacking AT1A receptors (KO) in renal proximal tubule (PT) have 10 mmHg lower baseline BP and less PT fluid reabsorption than wild type (WT). We tested the hypothesis that the lower BP is associated with less abundant renal Na transporters or regulators. Homogenates of renal cortex and medulla (n=6/group) were prepared and 1 and 1/2 protein amounts of each subjected to immunoblot analysis with specific antibodies and quantitated. Results for cortex and medulla, displayed as mean +/- SEM, normalized to mean abundance of WT=1 (*p <0.05), are summarized in figures. In KO vs. WT abundance of PT NHE3, the associated motor myosin VI, the paracellular NaCl transporter claudin 2, and the Na-HCO3 transporter NBCe1 are lower in KO; in the thick ascending limb (TAL) NKCC2 and its associated kinase SPAK are less abundant, and there is a tendency for lower DCT NCC and CCD ENaC in KO. The results support our hypothesis and suggest that KO of PT AT1R reduces transport routes not only in the PT but beyond the PT, in spite of increased volume flow from PT and lower BP.
Abstract P273: Increased Inhibition of Potassium Channel Currents by Angiotensin II in Sympathetic Neurons may Contribute to a Sustained Blood Pressure Elevation in (mRen2)27 Rats.
Authors: Sidach Serguei S , Pulgar Victor M , Aileru Azeez A
Published: June 2021

It is well established that the increased sympathetic tone may contribute to initiation and progression of various forms of hypertension. Several lines of evidence suggest a link between the renin-angiotensin system and sympathetic nerve activity in hypertension, and the previous studies in animal models demonstrated increased sympathetic output in the presence of Angiotensin II (AngII). To elucidate potential underlying molecular mechanisms of such phenomenon, we compared the effect of AngII on the whole-cell potassium channel currents in superior cervical ganglia (SCG) neurons isolated from hypertensive (mRen2)27 rats with overexpression of renin gene, and control Sprague Dawley® (SD) rats. In both groups, the whole-cell potassium channel currents were identified as rapidly-activating, 4-Aminopyridine-sensitive transient A-type currents, as well as slowly-activating tetraethylammonium-sensitive delayed rectifier currents. When the cell membrane was depolarized to -40, -30 and -20 mV from a holding potential of -80 mV, AngII (100 nM) profoundly inhibited A-type current, but the magnitude of such inhibition was not significantly different between neurons isolated from (mRen2)27 (38.1±6.2%, 47.8±5.7% and 52.1±5.7%; n=11) and SD rats (37.2±4.6%, 44.±4.5% and 46.1±4.8%; n = 13). Delayed rectifier potassium channel currents were isolated by holding cells at -40 mV, which resulted in complete elimination of the transient A-type current. In contrast to transient current, inhibition of the delayed rectifier current by AngII in the range of membrane potentials between +20 and +80 mV was significantly greater (p<0.05) in neurons obtained from (mRen2)27 rats (11.0±3.2% to 25.0±2.9%, n=12) when compared to SD rats (4.7±1.5% to 16.3±2.7%, n = 12). In both groups, inhibition of both channel types was completely abolished by 10 uM Losartan, indicating involvement of AT1 receptors. Our results suggest that in (mRen2)27 hypertensive rats, the increased inhibitory effect of AngII on delayed rectifier potassium channel currents could possibly lead to lowering spike threshold, which, in turn, could elevate sympathetic outflow and lead to sustained blood pressure elevation.
Abstract P274: Fruit Extract (blueberry, Cranberry, And Promegranate) Improved Insulin Resistance In Overweight Hypertensive And Normotensive Subjects
Authors: Novaes Ludmila N , Moraes Mariele , Katayama Keyla , Sangaleti Carine , Irigoyen Maria Claudia , Bortolotto Luiz , Lopes Heno
Published: June 2021

Arterial hypertension is frequently associated to glucose and lipid metabolism abnormalities. The purpose of this study was to determine if antioxidants (fruit extract) supplementation interfere with glucose and lipid metabolism in overweight hypertensive patients. A randomized clinical trial was conducted with 30 individuals, 23 hypertensive patients (group A) and 7 normotensive controls (group B). They were randomized to take 3 capsules of different fruits extract a day (blueberry, cranberry and pomegranate) or placebo for 4 weeks. This is a crossover study, which started with placebo changed to capsules and vice versa. Blood samples were collected after 12 hours fasting for biochemical tests (glucose, insulin, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides), anthropometric assessment (weight, height, and body mass index), systolic BP, diastolic BP and heart rate were evaluated at baseline, after 4, and 8 weeks. The comparisons between groups were held with the GLM repeated measures. Twenty three hypertensive patients (age 47 years, 14 females) and 7 normotensive controls (age 40 years, 7 females) were evaluated. BMI, blood pressure, heart and lipid profile did not differ between groups. HOMAir decreased significantly in both groups. See results in table 1.

Values are expressed as medians (±SD)

In these preliminary results a 4-weeks supplementation of antioxidants (fruit extract) improved insulin resistance in overweight hypertensive and normotensive subjects.

Financial support: FAPESP 2014/25808-3