bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P082: Elevated Circulating Endothelial-Derived Microvesicles Are Associated With Obesity-Related Endothelial Dysfunction
Authors: Brewster L. Madden , Bammert Tyler D , Hijmans Jamie G , Dow Caitlin A , Goulding Anabel , Greiner Jared J , Stauffer Brian L , Desouza Christopher A
Published: September 2020

Background:
Obesity reduces the number of years lived free of cardiovascular disease (CVD) and increases the number of years lived with CVD, contributing to increased morbidity and mortality risk across all ages. Experimental and clinical studies indicate that a proatherogenic endothelial phenotype is a major consequence of increased adiposity and a primary mechanism underlying obesity-related CVD. Extracellular microvesicles, particularly endothelial cell-derived microvesicles (EMVs), are seminal functional modulators of vascular health and disease. The purpose of this study was to determine: 1) if circulating EMV levels are elevated with obesity, independent of other cardiometabolic risk factors; and if so, 2) whether circulating EMVs are associated with obesity-related endothelial vasodilator dysfunction.

Methods:
Thirty sedentary, middle-aged adults (45-63 years) were studied: 15 normal weight (10M/5F; age: 56±1 yr; BMI: 23.2±0.4 kg/m2; body fat: 25.1±2.4 %) and 15 obese (10M/5F; age: 54±1 yr; BMI: 31.5±0.3 kg/m2; body fat: 37.2±1.7 %). All subjects were free of other cardiometabolic risk factors and overt disease. EMV identification (CD31
+
/42b
-
) and concentration in peripheral blood were determined by flow cytometry. Forearm blood flow (FBF: via plethysmography) was assessed in response to intra-arterial infusions of acetylcholine (4.0, 8.0 and 16.0 μg/100 mL tissue/min) and sodium nitroprusside (1.0, 2.0 and 4.0 μg/100 mL tissue/min).

Results:
Circulating EMV levels were ~100% higher (P<0.05) in obese (182±14 EMV/μL) compared with normal weight (91±12 EMV/μL) adults. FBF response to acetylcholine was significantly lower (~35%) in the obese (from 4.0±0.2 to 10.3±0.6 mL/100 mL tissue/min vs 4.3±0.3 to 15.4±0.8 mL/100 mL tissue/min) group. Circulating EMVs were significantly and inversely associated with total FBF response to acetylcholine (r=-0.55).

Conclusions:
Obesity, independent of other cardiometabolic risk factors, is associated with elevated circulating levels of EMVs. Higher circulating EMVs in obese adults may contribute to adiposity-related endothelial dysfunction and vascular disease risk. Indeed, circulating EMVs have been linked to disease risk, severity and outcome in other high-risk populations.
Abstract P083: Fetuin-a Induces Vascular Dysfunction Through Toll-like Receptor 4 And Nox1/4 Activation
Authors: Neves Karla B , Lopes Rheure A , Strembitska Anastasiya , Hepburn Ross , Beattie Wendy , Graham Delyth , Montezano Augusto C
Published: September 2020

Although studies demonstrate an important role for fetuin-A (FetA) in the inhibition of vascular calcification, convincing evidence suggests that fetuin-A is also involved in insulin resistance, inflammation and cardiovascular damage. The present study seeks to unravel FetA vascular effects and associated molecular mechanisms, focusing on oxidative stress and toll-like receptor 4 (TLR4). Vascular function studies were performed in mesenteric resistance arteries from WKY rats, wild-type, Nox1 KO, Nox4 KO and Ang II-dependent hypertensive mice (LinA3) and rat aortic endothelial cells (RAEC). ROS production (chemiluminescence, Amplex Red, ELISA) and pro-inflammatory markers expression (RT-PCR) were measured in VSMCs from WKY rats and RAEC. FetA impaired endothelium-dependent (LogEC50 7.320±0.08 M vs control 8.025±0.06) and endothelium-independent vasorelaxation (LogEC50 6.48±0.19 M vs control 7.38±0.12), p<0.05; effects blocked by tempol (superoxide dismutase mimetic), Nox1 inhibitor, ML171, and TLR4 inhibitor, CLI095. We did not observe any changes in contraction. FetA increased ROS production (62%) and peroxynitrite levels (158%) in VSMCs; while in RAEC, FetA increased ROS production (105%) followed by a decrease in H2O2 (62%) levels (p<0.05 vs control). FetA-induced effects on ROS were inhibited by ML171 and GKT137831 (Nox1/Nox4 inhibitor), as well as CLI095. Vascular dysfunction in arteries from Nox1 and Nox4 KO mice was unaffected by FetA. Activation of the FetA/TLR4/Nox axis led to an increase in IL-1β (190%), Il-6 (124%) and RANTES mRNA expression(116%) in RAEC, p<0.05 vs control. FetA enhanced vascular dysfunctionin LinA3 mice. Together, these results suggest that FetA through TLR4/Nox1 and 4-derived ROS leads to vascular dysfunction and inflammation, which may play an important role in the development of vascular injury during hypertension.
Abstract P087: Mitochondrial Reactive Oxygen Species Mediate Endothelial Glycocalyx Damage And Vascular Dysfunction In Hemorrhagic Shock And Resuscitation
Authors: Abdullah Sarah , Karim Mardeen S. , Legendre Mark , Rodriguez Laura , Friedman Jessica , Taghavi Sharven , Guidry Chrissy , Duchesne Juan , Jackson-weaver Olan
Published: September 2020

The endothelial glycocalyx forms an anti-thrombotic layer on the apical surface of endothelial cells and maintains the selective permeability barrier of blood vessels. Ischemia reperfusion injury like hemorrhagic shock is shown to cause glycocalyx damage. We have previously shown that mitochondrial reactive oxygen species (mitoROS) mediate glycocalyx damage in cultured endothelial cells. Of note, angiotensin II elevates endothelial mitoROS, suggesting a possible exacerbation of glycocalyx damage in hypertensive patients. It is unknown, however, whether mitoROS mediate glycocalyx damage
in vivo
. We hypothesize that mitoROS mediate glycocalyx damage in a rat model of hemorrhagic shock.We investigated the effect of mitochondrial ROS on the endothelial glycocalyx
in vivo
, in a rat model of hemorrhagic shock. In anesthetized rats, mean arterial pressure was reduced to 40 mmHg by withdrawing blood from the femoral artery and kept at 40 mmHg for 30 minutes. The rats were then resuscitated with IV (jugular vein) Ringer’s lactate solution for 30 minutes more. Sham rats received the vascular lines only. Syndecan-1 in the plasma was increased after 30 minutes of resuscitation with LR in hemorrhage/resuscitation (H/R) rats (p=0.02) but was not elevated in Sham rats (p=0.52). Resuscitation with IV mitoROS scavenger mitoTEMPOL significantly blunted this increase (5.9 pg/ml vs. 8.7 pg/ml, p=0.03). At the organ level, the glycocalyx was decreased in the endothelium of muscle (p=0.0444) and intestine (P=5.47 * 10
-7
) vascular beds in H/R rats vs. Sham rats. Our findings show that mitoROS mediate the glycocalyx damage after H/R. This mechanism suggests possible therapies that target mitoROS generation. Future work will investigate whether preexisting hypertension increases glycocalyx damage during H/R due to exacerbated mitoROS levels.
Abstract P088: The Gut Microbiome Contributes To The Cerebral Small Vessel Disease Phenotype In Spontaneously Hypertensive Stroke Prone Rats
Authors: Bryan Robert M , Phillips Sharon C , Durgan David J
Published: September 2020

In recent years, it has become apparent that the gut microbiome can influence the functioning and pathological states of organs and systems throughout the body. In this study we tested the hypothesis that the gut microbiome has a major role in the cascade of pathological events, including inflammation and hypertension, leading to the onset of cerebral small vessel disease (CSVD). To test this hypothesis, we used an animal model for hypertensive CSVD, the spontaneously hypertensive stroke prone rat (SHRSP). At birth, SHRSP pups were placed with foster dams of the SHRSP strain or dams of the WKY strain, the control strain that does not develop hypertensive CSVD. Similarly, WKY pups were placed with dams of the same or opposite strain. The rationale for cross fostering is that gut microbiomes are shaped by environmental bacteria of the foster dam and the nesting surroundings. Analysis of the bacterial genera in feces using 16S rRNA analysis demonstrated that the gut microbiome in the rat pups was strongly influenced by the foster dam. SHRSP offspring fostered on WKY dams had systolic blood pressures (SBPs) that were significantly decreased by 26 mmHg (P<0.001, N=5-7/group) at 20 weeks, compared to SHRSP offspring fostered on SHRSP dams. Similarly, WKY offspring fostered on SHRSP dams had significantly increased SBP compared to WKY offspring fostered on WKY dams, although the magnitude of SBP change was not as robust (9 mmHg, P<0.05, N=6-8/group). At ~20 weeks of age, rats fostered on SHRSP dams showed increased expression of IL-1a, TLR-2, E-CAD, Muc-2, and Il-17a in ileum regardless of the strain of the offspring (N=6/group, P<0.05). Loss of blood-brain barrier (BBB) integrity, an early marker of CSVD onset, was assessed by extravasation of IgG, which is confined to the plasma space under normal conditions. Extravasation of IgG was increased four-fold in SHRSP fostered on SHRSP dams compared to WKY rats fostered on WKY dams; however, extravasation of IgG was decreased in SHRSP fostered on WKY dams by ~ 50% (P=0.005, N=5/group). These findings demonstrate that although SHRSP is a genetic model for CSVD, the makeup of the gut microbiota has a major influence on gut and brain physiology, and ultimately in shaping the phenotype and onset of CSVD.
Abstract P091: New Rho-kinase Inhibitors Reduce Cardiac Dysfunction And Vascular Remodeling In Pulmonary Hypertension In Rats
Authors: Montagnoli Tadeu L , Da Silva Jaqueline S , Rocha Bruna S , Silva Marina , Santos-Carlos Bianca , Fraga Carlos , Zapata-Sudo Gisele
Published: September 2020

Pulmonary hypertension (PH) is characterized by extensive pulmonary vascular remodeling, leading to right ventricle (RV) hypertrophy and dysfunction. Since Rho kinases (ROCK) play a key role in smooth muscle proliferation and cardiomyocyte hypertrophy, this work evaluated the effects of ROCK inhibition by LASSBio-2020 and LASSBio-2065 on monocrotaline (MCT)-induced PH. After single injection of MCT (60 mg/kg i.p.), male Wistar rats were randomly divided in groups and treated with vehicle, LASSBio-2020 or LASSBio-2065 (60 μmol/kg/day, i.p.). Table 1 shows summarized data. After 14 days, ROCK inhibitors reduced pulmonary vascular resistance, indicated by the ratio of pulmonary acceleration time and ejection time (PAT/TET; p< 0.05) and medial wall thickness of distal pulmonary arterioles (p< 0.05). LASSBio-2020 and LASSBio-2065 also reduced Fulton index of RV hypertrophy (p< 0.05) and RV afterload, as shown by recovery of RV cardiac output (p< 0.05) and arterial elastance (p< 0.05). ROCK inhibitors improved diastolic function since both compounds reduced RV end-diastolic pressures (RVEDP) and Tau, measured through catheterization. Therefore, ROCK inhibition by LASSBio-2020 and LASSBio-2065 reverted functional and morphological alterations in PH rats and may represent a useful alternative for management of PH-associated RV dysfunction.
Abstract P092: Palmitoylation Controls Cell Surface Abundance Of Trpm7
Authors: GAO xing , Rios Francisco , Montezano Augusto C , Touyz Rhian M , Fuller William
Published: September 2020

Magnesium regulates numerous cellular functions and enzymes and abnormal magnesium homeostasis contributes to vascular dysfunction and the development of hypertension. The transient receptor potential melastatin 7 (TrpM7) has emerged as a key player in cardiovascular magnesium homeostasis. This bifunctional channel/kinase is ubiquitously expressed and regulates embryonic development. Its integral membrane ion channel domain regulates transmembrane movement of divalent cations, primarily Ca
2+
, Mg
2+
and Zn
2+
, and its kinase domain controls gene expression via histone phosphorylation. TrpM7 not only localizes on the cell surface to serve as a critical regulator of transmembrane Mg
2+
flux, but also forms an intracellular Zn
2+
release channel in vesicles of unknown origin. Palmitoylation is a dynamic reversible posttranslational modification, which regulates ion channel activity, stability, and subcellular localization. We found that TrpM7 is palmitoylated in multiple cell types. Here we sought to identify palmitoylated cysteines and the functional consequences of TrpM7 palmitoylation in HEK cells. Mutation of Cysteines 1143, 1144 and 1146 on TrpM7 (TrpM7-AAA) to alanines reduced its palmitoylation by 68.4±8% (n=13;
P
<0.05), identifying this cluster of cysteines as the principal sites of palmitoylation. Fluorescent microscopy indicated that TrpM7-AAA was retained in the endoplasmic reticulum (ER), but when the palmitoylated cysteines in TrpM7 were replaced with the analogous regions of TrpM2 (M7/M2) or TrpM5 (M7/M5), the chimaeric proteins could exit the ER despite being 80.6±13% (n=3;
P
<0.05) and 70.6±17% (n=3;
P
<0.05) less palmitoylated than wild type respectively. Using membrane-impermeable biotinylation reagents we found that palmitoylation alters the balance of distribution of TrpM7 between vesicular and cell surface pools. The proportion of M7/M2 and M7/M5 reaching the cell surface membrane was decreased by 69.0±14% (n=3;
P
<0.05) and 51.4±9% (n=3;
P
<0.05) respectively, compared to wild type. Therefore, inhibiting TrpM7 palmitoylation reduced its cell surface abundance. The impact of palmitoylation on TrpM7 channel activity and its contributions to hypertension and vascular pathologies need further investigation.
Abstract P093: Cytochrome B5 Reductase 3 And Xanthine Oxidase Coordinately Regulate Soluble Guanylyl Cyclase Physiological Redox State
Authors: Durgin Brittany G , Schmidt Heidi M , Hahn Scott A , Straub Adam C
Published: September 2020

In cardiovascular disease, oxidative stress can drive soluble guanylyl cyclase (sGC) heme oxidation resulting in the loss of the sGC heme (apo-sGC), the impairment of nitric oxide (NO) binding and cGMP production, and vasoconstriction. Consequently, a new class of therapeutic compounds sGC activators have been developed which target oxidized and apo-sGC to cause irreversible, NO-independent reactivation of cGMP production and vasodilation. While sGC activators have had varied clinical success, surprisingly few studies have defined the impact of NO-independent sGC activation on vascular physiology in healthy conditions. We found mesenteric and pulmonary arteries are two log orders more sensitive to NO-independent sGC activator BAY 58-2667 induced vasodilation than aorta; no difference in NO-dependent sGC vasodilation between vessels was observed. These data indicate the presence of an activatable physiological pool of oxidized and/or apo-sGC in pulmonary and mesenteric arteries. We recently published that smooth muscle cell cytochrome b5 reductase 3 (CYB5R3) acts to reduce oxidized heme sGC back to its NO-sensitive reduced heme state during vascular disease. We found transgenic CYB5R3 overexpression (CYB5R3 OE) mice were more resistant to BAY 58-2667 mesenteric artery vasodilation and blood pressure lowering compared to wild-type controls (n=5-9) under physiologic conditions. Also, healthy CYB5R3 OE pulmonary arteries had a near complete loss of BAY 58-2667 vasodilation suggesting both mesenteric and pulmonary arteries contain a pool of oxidized sGC. We next asked if physiological H
2
O
2
production accounts for changes in BAY 58-2667 responsiveness. We found using mitochondrial-specific catalase overexpression mice, that BAY 58-2667 vasodilation did not differ from controls in any vascular bed (n=4-6). We next tested whether xanthine oxidase (XO), which can produce H
2
O
2
at the endothelial cell surface of vessels, can impact physiological BAY 58-2667 vasodilation. We found that Febuxostat, a XO inhibitor, led to a significant decrease in mesenteric artery BAY 58-2667 induced vasodilation from ~70% to ~30% (n=6). Combined, these data provide evidence for CYB5R3 and XO as regulators of physiological sGC resistance artery vasodilation.
Abstract P094: Crosstalk Between Mas And ET
B
R Elicits Protective Effects In Endothelial Cells And Vascular Function
Authors: Kuriakose Jithin , Montezano Augusto C , Hood Katie Y , Alves-lopes Rheure , Sin Angie , Passaglia Patricia , Findlay Jane , Basheer Sehrish , Yusuf Hiba , Hepburn Ross , Santos Robson , MacLean Margaret , Baillie George , Touyz Rhian M
Published: September 2020

Mas and ET
B
receptors physically interact in endothelial cells (ECs) and are involved in the protective actions of angiotensin 1-7 (Ang (1-7)). We assessed whether the MAS/ET
B
R interaction plays a role in EC signalling and whether strategies to enhance MAS/ET
B
R association influence vascular responses. Human ECs were stimulated with Ang (1-7) (10
-7
M) in the presence/absence of A779 (Mas receptor antagonist, 10
-5
M) and BQ788 (ET
B
R antagonist, 10
-5
M). Protein expression and signalling activation were assessed by immunoblotting. NO production was evaluated by DAF-FM fluorescence and ROS production by chemiluminescence (superoxide anion) or amplex red (hydrogen peroxide (H
2
O
2
)). mRNA expression was assessed by qPCR. Endothelial function was assessed in mouse intact arteries by myography. Ang (1-7), through Mas and ET
B
R induced phosphorylation of eNOS (35%); followed by an increase in NO production (2.0 fold) (p<0.05 vs ctl). High throughput screening of protein:protein interaction compounds in an in-house library identified 23 potential enhancers of the MAS/ET
B
R interaction. Fluorescence polarization assays were used to further select the most potent enhancers and define their working concentration for testing in ECs (Enh1-4: 10
-5
M). Enh4 increased superoxide anion (55.6±26.3% vs ctl, p<0.05) and H
2
O
2
production (54.7±11.1% vs ctl, p<0.05), while Enh3 increased H
2
O
2
generation (48.1±15.4% vs ctl, p<0.05) in ECs. Moreover, Enh4 increased
Nrf2
(3.0 fold),
Sod1
(2.0 fold) and
Nqo1
(3.1 fold) mRNA expression (p<0.05 vs ctl). Enh3 and Enh4 increased NO production (Enh3: 21.2±7.4%; Enh4: 23.6±8.2% vs veh, p<0.05) in ECs. Acetylcholine (Ach) curves were performed to assess endothelium-dependent relaxation in the absence and presence of enhancers. Enh4 increased ACh-induced relaxation (Emax%: 96.7±4.6 vs ctl: 70.4±3.3, p<0.05), while other enhancers did not improve endothelial function. Taken together, increasing MAS/ET
B
R interaction with specific enhancers augments protective signalling in ECs and promotes endothelial-dependent vasorelaxation, particularly with Enh4. In conclusion, enhancing interactions between MasR and ET
B
R may be a new vasoprotective strategy to improve vascular function in cardiovascular disease.
Abstract P096: Alterations In Vascular
Rgs5
Expression In Erythropoietin-induced Hypertension Mediated By Erk1/2 Pathway Activation
Authors: Kumar Nitin , Hunker Kristina , Birt Isabelle , Ganesh Santhi
Published: September 2020

Introduction:
Erythropoietin (EPO)-induced hypertension is a common adverse event among patients receiving EPO therapy and is associated with risk of myocardial infarction and stroke, yet the underlying molecular mechanisms responsible for EPO related cardiovascular disease mortality are not clear. Regulator of G-protein signaling-5 (
Rgs5
) is highly expressed in vascular smooth muscle cells (SMCs) and its action in fine tuning G-protein signaling is critical in regulating vascular tone. Whether
Rgs5
plays a role in EPO-mediated hemodynamic changes is not known. We have previously shown that increased arterial pressure after EPO administration in mice is associated with significant reduction in aortic
Rgs5
expression. One of the proposed mechanisms of
Rgs5
mediated cellular response involves regulation of MAPK/ERK signaling pathway. Here, we investigated the role of
Rgs5
and ERK1/2 pathway signaling in EPO-induced hypertension in mice.

Methods:
10-12 weeks old male and female C57BL/6 mice were randomly divided into two groups: (1) Vehicle (0.9% saline-VEH), (2) EPO, (N=8). VEH and EPO were administered intraperitoneally (EPO 75U/30g, 3 times/week) for 20 days.
Rgs5
silencing was performed in mouse aortic SMCs (N=3), and phophoryated-ERK1/2 (p-ERK1/2) was quantified by western blot.

Results:
EPO treated mice showed increased p-ERK1/2 activity in aorta and heart (fold change in aorta = 1.37,
P
= 0.001; fold change in heart = 1.72,
P
= 0.015) suggesting MAPK activation as possible mediator of EPO-induced increase in blood-pressure.
In vitro
, EPO increased the p-ERK activity in mouse aortic SMCs (MOVAS VEH 0.99 ± 0.05, EPO 1.31 ± 0.13,
P
=0.032) and interestingly
Rgs5
silencing in MOVAS exacerbated EPO-induced p-ERK activation (
Rgs5
siRNA + EPO 1.70 ± 0.11,
P
=0.038 vs EPO), suggesting a potential role of
Rgs5
in EPO mediated ERK activation and thus in regulating vascular tone.

Conclusions:
Our data indicated that
Rgs5
is crucial in regulating EPO-induced ERK1/2 activation and that
Rgs5
and its signaling pathway may serve as novel treatment targets in reducing cardiovascular complications in patients receiving EPO therapy.
Abstract P097: Angiotensin Receptor Neprilysin Inhibition Improves Arterial Stiffness And Endothelial Function In Heart Failure With Reduced Ejection Fraction
Authors: Nathaniel Sangeetha D , McGinty Shane , Edwards David G , Farquhar William B , Witman Melissa A , Hosmane Vinay , Wenner Megan M
Published: September 2020

The mechanisms for the benefits of Angiotensin Receptor Neprilysin inhibitor (ARNi) in heart failure patients with reduced ejection fraction (HFrEF) are likely beyond blood pressure (BP) reduction. Vascular function, a prognostic marker in HFrEF, improves with ARNi in animal models. Improvements in vascular function may contribute to benefits from ARNi in HFrEF; however, this has yet to be demonstrated in humans. The purpose of the study was to test the hypothesis that arterial stiffness and endothelial function would improve after 12 weeks of ARNi in HFrEF.

Methods:
HFrEF participants with NYHA class II-III were enrolled from local cardiology clinics and completed experimental visits at baseline and 12 weeks later: 13 participants were prescribed ARNi by their cardiologist [62±10 years, Men: 10, BMI: 30±5 kg/m
2
, EF: 28±6 %; Non-ischemic cardiomyopathy (NICM): 8], 10 participants continued on conventional treatment [CON: 60±7 years, Men: 6, BMI: 31±6 kg/m
2
, EF: 31±5 % and NICM: 4; all P=NS]. During each experimental visit, arterial stiffness was assessed via carotid-femoral pulse wave velocity (PWV; Sphygmocor PVx system) and endothelial function by brachial artery flow-mediated dilation (FMD) using standard techniques. Statistical analyses were performed using 2x2 repeated-measures ANOVA.

Results:
Baseline mean BP (MAP) was similar between ARNi (93±14 mmHg) and CON (85 ± 10 mmHg; P=0.13); MAP tended to decrease after 12 weeks of ARNi (88 ± 11 mmHg; P=0.08) but not CON (90 ± 17 mmHg; P=0.14) (ANOVA interaction P=0.03). PWV tended to be higher at baseline in ARNi (8.8 ± 2.5 m/s) compared to CON (7.0 ± 2.5 m/s; P=0.09); PWV decreased after 12 weeks of ARNi (7.0 ± 1.7 m/s; P<0.01) and was unchanged in CON (7.4 ± 2.4 m/s; P=0.33) (ANOVA interaction P<0.01). When controlling for MAP, the effect of ARNi on PWV remained (P<0.01). At baseline, FMD was similar between ARNi (2.81 ± 2.05%) and CON (4.75 ± 3.75%; P=0.13); however, FMD increased after 12 weeks of ARNi (5.73 ± 1.87%; P<0.001) but not in CON (5.37 ± 3.38%; P=0.33) (ANOVA time P<0.001, interaction P=0.01).

Conclusion:
ARNi improves arterial stiffness and endothelial function in HFrEF. Understanding the mechanisms of ARNi in HFrEF is crucial as it may pave the way for better interventions in other cardiovascular diseases.
Abstract P099: Loss Of The Brown-Like Phenotype Of Aortic PVAT Is Associated With Mitochondrial Dysfunction In Obesity-Induced Endothelial Dysfunction In Female Rats
Authors: Maddie Nicole , Carrillo-sepulveda Maria Alicia A
Published: September 2020

Endothelial dysfunction is a major complication of obesity and is an early contributor to hypertension. Perivascular adipose tissue (PVAT) surrounds most blood vessels and has different phenotypic properties based on its anatomical location. Thoracic aortic PVAT from humans and rodents is a brown-like adipose tissue and plays a vasculo-protective role under physiological conditions. In obesity, aortic PVAT expands, switches from a brown-like to a white-like phenotype and contributes to endothelial dysfunction. We hypothesized that loss of the brown-like phenotype of aortic PVAT in obesity is associated with mitochondrial dysfunction, resulting in PVAT and endothelial dysfunction. Eight-week-old female Wistar rats were randomized into two experimental groups: the Lean group (n=8) received a chow diet (5% fat, 48.7% carbohydrate [3.2% sucrose], 24.1% protein) and the Obese group (n=8) received a western diet (21% fat, 50% carbohydrate [34% sucrose], 20% protein), for 20 weeks. Increased body weight (340.57 vs. 265.37g leans, p<0.05) was confirmed in the obese group. At the experimental endpoint, thoracic aortas with intact (+PVAT) or removed PVAT (-PVAT) were obtained for analysis. Endothelial function was assessed in aortic rings +PVAT or -PVAT by performing concentration-response to acetylcholine using wire myography. The aortic ring (-PVAT) from the obese group exhibited impaired endothelium-dependent vasodilation (p<0.01). This effect was heightened in aortic rings (+PVAT) (p<0.05), showing a negative effect of PVAT on endothelial function during obesity. Mitochondrial dysfunction in PVAT from the obese group was characterized by decreased mitochondrial density (30% reduction, p<0.05), detected by quantification of Mitotracker fluorescence, and increased reactive oxygen species levels (4.34-fold increase, p<0.01), as evidenced by DHE staining. These effects were accompanied by decreased uncoupling protein-1 expression in the obese group (55% reduction, p<0.01). Moreover, Oil Red O staining showed larger lipid droplets in aortic PVAT from the obese group. Our results support that obesity-induced endothelial dysfunction is associated with a loss of the brown-like phenotype and mitochondrial dysfunction in PVAT.
Abstract P100: Opioids Cause Sex-Specific Vascular Remodeling Via Cofilin-ERK Signaling: Female Mice Present Higher Risk Of Developing Morphine-Induced Vascular Dysfunction Than Male Mice
Authors: Cheon Soyoung , Edwards Jonnelle M , Tomcho Jeremy C , Bearss Nicole R , Joe Bina , McCarthy Cameron G , Wenceslau Camilla F
Published: September 2020

Recent studies have shown that chronic use of prescription opioids leads to increased risk of cardiovascular events and pulmonary arterial hypertension. Indices of vascular age and arterial stiffness are increased in opioid dependent patients and the effects are more marked in women. We hypothesized that chronic exposure to exogenous opioids causes sex-specific vascular remodeling via suppression of actin depolymerization. Using culture pressure myographs, we performed continuous intraluminal infusion of morphine (10μM for 24 hours) in pressurized mesenteric resistance arteries (MRA, fifth order) from male and female mice (C57BL/6NTac, 10 weeks old; n=4). Intraluminal pressure curve showed that morphine reduced lumen diameter by 23% (without morphine: 185 ± 10μm vs. morphine: 143 ± 14μm*) in MRA from female mice, whereas little to no change was observed in MRA from male mice (Fig. 1). Cofilin is a key molecule in preventing stress fiber overassembly and is inactivated by extracellular signal-regulated kinases (ERK). Here, we observed that after 24-hour exposure, morphine (10μM) phosphorylated and inactivated cofilin 1.6 fold [AU: control: 0.59 ± 0.14 vs. morphine: 0.95 ± 0.10*, *p<0.05, t-test] and increased ERK phosphorylation 1.8 fold (AU: control: 0.51 ± 0.11 vs. morphine: 0.90 ± 0.13*, *p<0.05) in MRA from female mice. On the other hand, morphine decreased phospho-ERK 1.5 fold (AU: control: 0.91 ± 0.08 vs. morphine: 0.59 ± 0.11*, *p<0.05) but had no effect on the levels of cofilin in MRA from male mice. These results have significant implications for the previously unexplored role of exogenous opioids as a modifiable cardiovascular risk factor, particularly in women.
Abstract P102: Generation Of A Novel Peptoid-peptide Analog Of Alpha-calcitonin Gene Related Peptide (α-CGRP) For The Treatment And Prevention Of Cardiovascular Diseases
Authors: Kumar Ambrish , Servoss Shannon , DiPette Donald J , Potts Jay D
Published: September 2020

Alpha-calcitonin gene related peptide (α-CGRP) is a 37-amino acid cardioprotective neuropeptide. Studies carried out in our laboratory and others establish α-CGRP as a potential therapeutic agent against a variety of cardiovascular diseases. However, the short half-life of α-CGRP limits its use in any long-term treatment regime. The goal of the present study is to develop an α-CGRP agonist analog with extended bioavailability using peptoid chemistry. A peptoid is a
N
-substituted glycine peptidomimetic molecule and is identical to α-amino acid except that the side chain in a peptoid is attached on the nitrogen rather than the α-carbon atom. Inclusion of a peptoid makes the native peptide protease-resistant and thus biostable
in vivo
. Using a solid-phase submonomer method, we synthesized a novel human α-CGRP analog containing two monomers of
N
-methoxyethylglycine (NMEG) peptoid at the N-terminus. Electrospray mass spectrometry (MALDI-TOF) analysis showed that the molecular mass of synthesized peptoid-peptide hybrid, NMEG-α-CGRP, was 4044 that is ~6.7% more than native peptide. An
in vitro
trypan blue cell exclusion assay demonstrated that incubation of NMEG-α-CGRP (5 μM) for 7 days did not affect the viability of rat H9C2 and mouse HL-1 cardiac cells. To evaluate the biological activity of NMEG-α-CGRP, a subcutaneous injection of human α-CGRP (10 μg/mice) and NMEG-α-CGRP (1, 3, 10, and 30 μg/mice) were given in 9-week-old C57BL6 mice (n=2 mice/dose), and blood pressure (BP) was measured using a tail-cuff method. A dose response curve showed that NMEG-α-CGRP decreased BP in mice in a time-dependent manner. Beginning with and injection of 3 μg of NMEG-α-CGRP, a dip in BP (85 ± 1 mmHg; in ±SD) was observed at 10 min after injection, and BP returned to baseline (125 mmHg) by 6 h, 18 h, and 24 h when injected with 3, 10, and 30 μg doses, respectively. Moreover, 10 μg of human-α-CGRP and NMEG-α-CGRP lowered BP from baseline for 2 h and 18 h, respectively, suggesting that NMEG addition increased stability, and thus bioavailability, of α-CGRP
in vivo
. In summary, our results show that a NMEG based α-CGRP modification is an effective approach to increase stability and, thus, bioavailability of α-CGRP
in vivo
making α-CGRP a viable therapeutic drug to treat cardiovascular diseases.
Abstract P104: Impact Of Orthostatic Hypotension In Inpatient Rehabilitation
Authors: Okamoto Luis E. , Sharma Purnima , Massey Lauren , Biaggioni Italo , Johns Jeff
Published: September 2020

Inpatient rehabilitation is crucial in the management of patients after injuries, surgery, or those with debilitating or neurological conditions. Orthostatic hypotension (OH) is commonly associated with these conditions, and therefore, the inability to stand and participate in rehabilitation therapy can negatively impact their recovery. To test this hypothesis, we study a cohort of 8352 patients who underwent rehabilitation at Vanderbilt Stallworth Rehabilitation Hospital between 2014-2019 to assess the prevalence of OH and its impact on the length of stay (LOS), functional independence measure (FIM) change (discharge FIM-admission FIM), and FIM efficiency (FIM change/LOS), compared to patients without OH admitted during the same period. We found that the prevalence of OH was 3.8% (n = 314; 60±18 yrs; 227 men). OH was commonly associated with patients admitted for neurological conditions (29%, n = 91), spinal cord dysfunction (22%, n = 71), orthopedic disorders (13%, n = 13), and stroke (10%, n = 33). On admission, patients with OH had a longer predicted LOS compared to those without OH (18±6
vs.
16±5 days, respectively,
p
= 0.001), reflecting greater medical complexity. Moreover, the actual LOS at discharge was significantly longer in OH patients compared to those without OH (19±12
vs.
13±9 days;
p
= 0.001) as was the difference between actual-predicted LOC (1±11
vs.
2±8;
p
= 0.001). The change in FIM was similar in both patients with and without OH (30±26
vs.
33±20, respectively;
p
= 0.52) but FIM efficiency was lower in OH patients (2±2.2
vs.
3±2.2 without OH;
p
= 0.001). OH prolonged LOS regardless of the admission diagnosis (stroke, other neurological conditions, spinal cord dysfunction and orthopedic disorders). In conclusion, OH is present in a significant proportion of patients undergoing acute rehabilitation and adversely impacts the recovery of these patients as measured by lower FIM efficacy and a prolonged rehabilitation period to achieve the same functional gain as patients without OH.
Abstract P109: Activation Of Hypothalamic Glutamate Receptors Is Required For Angiotensin II-elicited Hypertensive Response Sensitization (HTRS) In Rats
Authors: Xue Baojian , Zhang Hua , Beltz Terry , Li De-Pei , Johnson Alan
Published: September 2020

Hypertension is associated with sympathetic nervous system activation, which involves interactions between the renin-angiotensin system and glutamatergic mechanisms in the hypothalamic paraventricular nucleus (PVN). Using the Induction-Delay-Expression (IND-DEL-EXP) experimental paradigm, our previous studies demonstrated that administration of a subpressor dose of angiotensin (ANG) II (10 ng/kg/min) during IND sensitizes subsequent ANG II (120 ng/kg/min)-elicited hypertension during EXP. Systemic or intracerebroventricular administration of glutamatergic NMDA-R antagonists (MK801 and AP5) during the IND period abolishes the IND of ANG II-elicited hypertensive response sensitization (HTRS). In the present study, we investigated further the role of hypothalamic glutamate and glutamate receptors (GluRs) in ANG II-elicited HTRS. First, PVN neurons to be studied
in vitro
were back-labelled by a tracer injected into the rostral ventrolateral medulla (RVLM). After sufficient time for retrograde transport, the rats were treated with a subpressor dose of ANG II sufficient to induce HTRS. In PVN brain slices studied following DEL, basal firing activities of PVN-RVLM projecting neurons did not differ between rats treated with saline and rats treated with a subpressor dose of ANG II. However, bath application of ANG II (2.0 μM) induced a significant increase in the firing rate of PVN-RVLM neurons in rats treated with ANG II during IND as compared to saline treated controls. This enhanced excitatory effect of ANG II on PVN-RVLM neurons was blocked by an NMDAR antagonist, AP5 (50 μM). Pre-treatment with the subpressor dose of ANG II during IND also significantly increased evoked NMDAR- excitatory postsynaptic currents in PVN-RVLM neurons. In functional studies, we found that bilateral PVN microinjections of glutamate (100 nm, 100 nl) during IND produced HTRS whereas PVN vehicle injections did not (Δ50.8±3.2 vs Δ20.8±6.5 mmHg). Taken together, our findings indicate that GluR activation in the PVN is sufficient and necessary for the IND of ANG II-elicited HTRS. The results indicate that a sustained increase in excitability of PVN-RVLM projecting neurons is likely to play an important role in maintaining the CNS state that produces HTRS.
Abstract P113: Genotypic Effect Of
Foxo3
On Lifespan Is Confined To Men With A Cardiometabolic Disorder (hypertension Or Coronary Heart Disease Or Diabetes)
Authors: Morris Brian J
Published: September 2020

FOXO3
is a prominent human longevity gene. To date, no-one has examined whether longevity-associated genetic variants of
FOXO3
protect against mortality in all individuals, or whether they protect against mortality only in individuals with aging-related diseases. Here, we examined this issue for the first time. We tested 9 representative longevity associated single nucleotide polymorphisms (SNPs) located within a haplotype block of
FOXO3
for association with mortality in 3,584 elderly American men of Japanese ancestry. At baseline (1991-1993), 2,512 had either diabetes (n=1,010), coronary heart disease (CHD; n=738), or hypertension (n=1,919), and 1,072 lacked any of these cardiometabolic disorders (CMDs). The men were followed from baseline until Dec 31, 2019. The longevity-associated (minor) alleles of all 9 SNPs were strongly associated with longer lifespan in individuals who had a CMD (covariate-adjusted hazard ratios [HRs] for each SNP: 0.82-0.95 [
P
=0.00008 to 0.020]; covariate adjusted haplotype analysis HR=0.81,
P
=0.0003). For just diabetes, just hypertension, and just CHD, haplotype HRs were 0.77, 0.82 and 0.83 (
P
=0.007, 0.0003 and 0.10), respectively. Reduced mortality was strongest for carriers of the minor (
G
) allele of SNP
rs2802292
(covariate adjusted HR=0.82, 95% CI 0.75-0.90;
P=
0.00008), as were survival curve differences (covariate adjusted Cox proportional hazard model difference,
P
=0.0002). As expected, men without a CMD outlived men with a CMD (Kaplan-Meier Log-rank chi-squared=24.8,
P
=0.0001). There was, however, no genotypic difference in lifespan of men who did not have a CMD (haplotype HR 1.02,
P
= 0.74). We propose that in individuals with a cardiometabolic disorder, longevity-associated genetic variants of
FOXO3
enhance resilience mechanisms in cells and tissues to help protect against cardiometabolic stress caused by CMDs, thereby allowing CMD-affected men to live longer than men without the protective variants.
Abstract P117: Blood Pressure Heterosis And Genotype-Specific Regulation Of
C17h6orf52
Authors: Clark Karen C , Wagner Valerie , Kwitek Anne E
Published: September 2020

Although blood pressure (BP) is highly heritable, the genetic etiology is complex, and causative genes do not completely explain observed variation. Using the Lyon Hypertensive (LH) rat—a well-characterized polygenic inbred model—we previously identified a novel candidate gene (
C17h6orf52
) as a master regulator of gene expression regulating cardiometabolic traits. CRISPR-Cas9 gene editing was used to generate a mutation in
C17h6orf52
, and blood pressure in homozygous wildtype (WT), heterozygous (HET) and homozygous mutant (MUT) females was characterized via radiotelemetry. Herein, we show evidence of BP heterosis (i.e. hybrid vigor) in females from the C17h6orf52 strain.

Systolic (SBP) and diastolic (DBP) pressures (mmHg) were determined in WT, HET, and MUT male and female rats on normal chow (CHOW), and 4% salt (NaCl) diet (to determine salt sensitivity). All measures are presented as mean±SEM, and significant results are indicated according to statistical test. No significant differences were identified in males, but the HET females (n=5) had significantly lower systolic blood pressure than either WT (n=7) or MUT (n=8) at both baseline and salt-stressed conditions:

SBP-CHOW: (WT: 144±3.28; HET: 136.0±0.83

*

; MUT: 142.6±2.13)DBP-CHOW: (WT: 98.3±2.29; HET: 94.0±0.68; MUT: 97.4±1.76)SBP-NaCl: (WT: 150.4±3.40; HET: 142.2±1.19

*

; MUT: 150.2±1.83)DBP-NaCl: (WT: 103.3±2.75; HET: 98.2±1.68; MUT: 103.1±1.68)

Tissues were collected to assess
C17h6orf52
expression relative to WT, displayed below as confidence intervals. While there were no differences between WT and MUT expression, liver
C17h6orf52
was significantly overexpressed in heterozygous females.

C17h6orf52
2^-ddCT: (WT: 0.91-1.09, n=8; HET: 1.33-1.73
$
, n=5; MUT: 0.85-1.19, n=8)

There were no striking differences in any metabolic phenotypes, such as body weight and adiposity, thus these data suggest that C17h6orf52 is protective against increases in blood pressure specifically. Continued study will attempt to tease apart allele-specific regulation and protein functions in heterozygous animals of both sexes.

*

p<0.05 by Kruskal-Wallis
$
p<0.05 by 1-WAY ANOVA
Abstract P129: Angiotensin II Does Not Directly Stimulate The TLR4-MD2-MyD88 Inflammatory Pathway
Authors: Okechukwu Charles C , Chappell Mark C
Published: September 2020

Current evidence suggests that the deleterious actions of Angiotensin II (Ang II) reflect activation of both innate and adaptive inflammatory pathways. Indeed, recent studies find that Ang II stimulates the TLR-4 signaling cascade to promote cytokine release and renal injury by directly binding to the TLR4 accessory protein MD2 to induce TLR-4 dimerization and internalization that is independent of the AT
1
receptor (AT
1
R). Moreover, knockdown of TLR4 or MD2 attenuates renal injury and expression of pro-inflammatory cytokines in a chronic Ang II infusion model and in rat proximal tubule NRK-52e cells treated with Ang II. In the present study, we evaluated the proposed Ang II-TLR4-MD2 pathway in the NRK-52e cells regarding the stimulation of the chemokine MCP-1 (CCL2) and activation of the TLR4-MyD88 complex. NRK-52e cells, maintained in antibiotic-free media, were transferred to serum-free media prior to stimulation with the TLR4 agonists LPS and palmitate or Ang II for 24 hrs; reported data are the means ± SEM, n=4. The NRK-52e cells were sensitive to a low dose of LPS (1 ng/ml) stimulating CCL2 release 20-fold [Basal: 24.3 ± 1.0 pg/ml vs. 504 ± 30.4 pg/ml, p<0.001]. The LPS induced release of CCL2 was abolished by the specific TLR4 inhibitor Tak-242 [TAK: 23.9 ± 1.3 pg/ml] and was significantly reduced by the MD2 inhibitor L48H37 [135 ± 24.2 pg/ml]. Palmitate [100 μM] also stimulated CCL2 release [482 ± 21.0 pg/ml; p<0.001] that was abolished by TAK [32.3 ± 6.3 pg/ml]. The overall effects are consistent with previously reported responses to LPS and palmitate in these cells, as well as TLR4 protein expression and TAK inhibition. However, Ang II treatment over a dose range of 0.1 to 10 μM for 24 hrs failed to elicit CCL2 release [24.3 ± 1.0, 22.6 ± 0.5, and 27.6 ± 2.3 pg/ml, respectively; P>0.1 vs. Basal]. Ang II [1 μM] also failed to augment the CCL2 response to LPS [496 ± 6.5 pg/ml] or palmitate [511 ± 35.0 pg/ml]. Finally, pre-treatment of the NRK-52e cells with the AT
1
R antagonist candesartan [5 μM] failed to attenuate CCL2 release to LPS [512 ± 21 pg/ml]. We conclude that Ang II does not directly stimulate the TLR4-MD2 complex to induce CCL2 and that the inflammatory events associated with Ang II
in vivo
may reflect the release of other factors (LPS, AGEs, HMGB1) that serve as TLR-4 agonists.
Abstract P130:
In Vitro
Treatment Of Isolated Renal T Cells With HMGB1-neutralizing Antibody Decreases Activation In Hypertensive Males And Females
Authors: Snyder Elizabeth , Sullivan Jennifer C
Published: September 2020

Background:
Renal necrosis induces the release of pro-inflammatory danger associated molecular patterns (DAMPs), and HMGB1 is the best-characterized DAMP. Previous studies published by our lab found that male spontaneously hypertensive rats (SHR) have greater renal necrosis and more pro-inflammatory T cells than females. The current study tested the hypothesis that greater HMGB1 in male SHR will result in greater renal T cell activation compared to female SHR.

Methods:
Male and female SHR were euthanized at 13 weeks of age. One kidney was sectioned and the cortex homogenized for HMGB1 quantification by ELISA (n=5 per group). The remaining kidney was homogenized under sterile conditions and renal T cells isolated. T cells were cultured with total splenocytes from sex-matched WKY. Additional experiments were conducted in the presence of HMGB1-neutralizing antibody (n=6) or control IgG antibody (n=4). After 72 hours of culture, CD71
+
cells were assessed by flow cytometric analysis as a measure of T cell activation and resulting data are expressed as a percentage of total T cells.

Results:
Renal HMGB1 levels were statistically comparable between male SHR and female SHR (1.61±0.1 vs. 1.3±0.1, respectively; p=0.12). Despite this, T cell activation was higher in male SHR than females (% CD71
+
cells: 92±0.3 vs. 70±2; p<0.0001). IgG antibody treatment did not alter T cell activation in either sex (% CD71
+
cells: 91±0.2 vs. 74±2), while treatment with HMGB1-neutralizing antibody decreased T cell activation in both sexes (% CD71
+
cells: male SHR, 79±1; female SHR, 57±1.6; 2-way ANOVA: p
sex
<0.0001, p
treatment
<0.0001, p
int
=0.49).

Conclusion:
Although male SHR did not have higher levels of HMGB1 than female SHR, they had significantly greater T cell activation compared to females. Treatment with HMGB1-neutralizing antibody decreased T cell activation in both males and females, but sex differences in T cell activation were maintained, suggesting that HMGB1 contributes to T cell activation equally in both sexes. Further studies will be required to determine the physiological basis of sex differences in renal inflammation.
Abstract P131: Jak2 Expression In Cd11c+ Myeloid Cells Plays A Role In Salt-sensitive Hypertension Through An Enac-dependent Mechanism.
Authors: Aden Luul A , Pitzer Ashley , Grimes Samantha , Ray Evan C , Kleyman Thomas R , Kirabo Annet
Published: September 2020

Hypertension is a major risk factor for development of cardiovascular disease. Excess dietary salt contributes to inflammation and the genesis of hypertension. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase-dependent formation of immunogenic isolevuglandin (IsoLG)-protein adducts leading to inflammation and salt-sensitive hypertension. We hypothesized that Jak2 expression, specifically in CD11c
+
myeloid cells, regulates expression of ENaCγ and promotes salt-sensitive hypertension. Our results indicate that deletion of Jak2 in CD11c
+
myeloid cells reduced the salt-induced expression of ENaCγ in CD11c+ cells. Moreover, mice lacking Jak2 in CD11c+ cells developed a blunted hypertensive response (123.8±4.7) during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride (L- NAME)/high salt model of salt-sensitive hypertension compared to their wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention (69.55±5.8) in response to high salt-feeding when compared to the wildtype litter mate controls (57.89±9.5). These results indicate that Jak2 expression in CD11c
+
myeloid cells plays a role in salt- sensitive hypertension through an ENaC-dependent mechanism.