bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 256: Loss-of-function Gln287 Variant in EPHX2 is Associated With Increased Insulin Sensitivity
Authors: Ramirez Claudia E , Shuey Megan M , Shibao Cyndya , Luther James M , Brown Nancy J
Published: March 2022

Epoxyeicosatrienoic acids (EETs) induce vasodilation via activation of calcium-sensitive potassium channels and smooth muscle cell hyperpolarization. Soluble epoxide hydrolase (sEH) hydrolyzes EETs to less biologically active metabolites. The variant Gln287 of
EPHX2
encodes for a sEH enzyme with reduced hydrolase activity. In murine models of type 2 diabetes, inhibition of sEH or
EPHX2
deletion results in improved insulin sensitivity. In humans, forearm blood flow is significantly increased in Gln287 variant allele carriers. Given that insulin sensitivity is influenced by vascular perfusion, we hypothesized that insulin sensitivity would be increased in Gln287-carriers compared with wild-type individuals. Insulin secretion and sensitivity were assessed in 63 volunteers (43 with metabolic syndrome, 34 females, 42.5 ± 13 years old, 15 black Americans) during hyperglycemic clamps. Statistical analyses were adjusted for BMI. Plasma total- (r=0.63, p=0.007), 14-15- (r=0.62, p=0.008), and 11-12- (r=0.57, p=0.02) EET concentrations correlated with insulin sensitivity. There was no effect of genotype on insulin secretion (not shown). In individuals without metabolic syndrome, insulin sensitivity was significantly increased in Gln287 variant carriers compared with wild-type individuals (Fig. A). Insulin sensitivity was decreased in individuals with the metabolic syndrome, but also trended higher in Gln287 variant carriers when compared with wild-type individuals (Fig. B). Taken together, these results suggest that the loss-of-function Gln287 variant of
EPHX2
is associated with increased insulin sensitivity.
Abstract 257: Mapping of Chromosome 2 Regions Linked to Vascular Inflammation using Congenic Rats
Authors: Rehman Asia , Yamamoto Naoki , Mian Muhammad Oneeb Rehman , Idris-Khodja Nourredine , Barhoumi Tlili , Kwitek Anne E. , Paradis Pierre , Schiffrin Ernesto L
Published: March 2022

Background:
Chromosome 2 introgression from normotensive Brown Norway rats (BN) into hypertensive Dahl salt sensitive (SS) background (consomic SB2) reduced vascular inflammation and restored Treg function. We hypothesized that the BN chromosome 2 contains genes that reduce vascular inflammation, which could be mapped using congenic rats containing different portions of BN chromosome 2 on the SS background.

Methods:
Twelve-to-13 week old male BN, SS, SB2, congenic (SB)A,

SBB and SBE rats fed normal salt diet were studied. Systolic blood pressure (SBP) was measured by telemetry. Aortic vascular cell adhesion molecule 1 (VCAM-1) and fibronectin expression, collagen content and reactive oxygen species (ROS) production were determined. Spleen Treg (CD4
+
CD25
hi
) and CD4
+
CD25
-
T lymphocytes were characterized and cultured. Transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-17 and IL-6 were measured in culture media.

Results:
SS, SB2 and SBE exhibited 20 mmHg higher SBP compared

to BN, SBA, and SBB (
P
<0.05). Aortic and perivascular ROS production and aortic VCAM-1 expression were ≥2.7- and ~1.5-fold greater, respectively, in SS and SBB (
P
<0.05) compared to BN, SB2, SBA and SBE. Aortic media collagen content was 3-fold greater in SS and 1.7-fold in SB2 and SBB (
P
<0.05) compared to BN, SBA and SBE. Aortic fibronectin expression was ~1.5-fold higher in SS and SBE (
P
<0.05) compared to SB2 and SBA or BN. The percentage of spleen CD4
+
CD25
-
cells was higher in SS, SB2 and SBE (~16%) compared to BN, SBA and SBB (~12%, <0.05). Treg percentage was lower in SBA (2%) compared to SS and SB2 (3%,
P
<0.05). CD4
+
CD25
-
from SS or SBB secreted less TNF-α, IFN-γ and IL-6 (≤141, 1428 and 13 pg/10
5
cells, respectively,
P
<0.05), compared to SB2 and SBE (~320, 2350 and 50 pg/10
5
cells, respectively) or BN (458, 3552 and 57 pg/10
5
cells, respectively). Treg IL-10 and IL-17 production was higher in SB2 (9597 pg/10
5
cells), and SS and SB2 (>90 pg/10
5
cells), respectively (
P
<0.05), compared to congenic rats (~2540 and 23 pg/10
5
cells, respectively) or BN (2497 and 9 pg/10
5
cells).

Conclusion:
Some genes regulating vascular injury and inflammatory responses are contained within the fragment of BN-chromosome 2 present in congenic SBA and SBE rats.
Abstract 267: Ablation of Sympathetic Neurons Projecting to Mesenteric Blood Vessels Attenuates Nicotine-Induced Rise in Systemic Arterial Pressure
Authors: Hammond Bradley D , Kreulen David L
Published: March 2022

Celiac ganglionectomy attenuates the increase in arterial pressure (AP) seen in the DOCA-salt model of hypertension; however, it is unknown which population of neurons in the celiac ganglion (CG) is responsible for this attenuation. Therefore, we ablated sympathetic post ganglionic neurons selectively innervating mesenteric blood vessels by applying a retrograde tracer, cholera toxin subunit b, conjugated to the neurotoxin saporin (CTB-SAP) on multiple, adjacent branches (~6mm) of mesenteric arteries and veins. Control animals received PBS instead of a CTB-SAP treatment. After 7-10 days rats were anesthetized, and fitted with a femoral catheter to record AP. Nicotine (200mM) was then applied to the surface of the surgically-exposed CG, which caused a significant increase in AP in control animals but not in CTB-SAP treated animals (
Table 1
). Quantitative morphometry done on the CG revealed a significant reduction in the density of ganglionic neurons in CTB-SAP treated animals when compared to control animals (
Table 1
). This study shows ablating CG neurons that innervate such a small portion of the mesenteric vasculature reduces the elevation of systemic AP induced by nicotine. Moreover, this study gives way for further investigations into how ablation of neurons innervating mesenteric arteries or veins can modulate AP.
Abstract 270: Exercise During Pregnancy Restores Angiogenic Balance But Does Not Lower Blood Pressure in Rupp Hypertensive Rats
Authors: Banek Christopher T , Needham Karen M , Gillham Haley E , Gilbert Jeffrey S
Published: March 2022

Although clinical studies suggest women continuing to exercise during pregnancy have a decreased incidence of preeclampsia, little is known regarding the effects of an exercise regimen started at the onset of pregnancy. Our laboratory has previously reported voluntary wheel running in rats for 6 weeks before and during (EBD) pregnancy increases circulating free vascular endothelial growth factor (VEGF), restores angiogenic potential, and attenuates placental ischemia induced hypertension. Our hypothesis was exercise during (ED) pregnancy without previous exercise training would decrease blood pressure and restore angiogenic potential in pregnant rats with reduced uterine perfusion pressure (RUPP)-induced hypertension. Rats were given free access to running wheels upon confirmation of pregnancy, and running distance was monitored weekly. RUPP (n = 10) or Sham (n= 10) surgery was performed on day 14 of gestation (term=21). Arterial pressure (AP) was measured via indwelling catheter in conscious rats and tissues were collected on day 19. Serum angiogenic potential was assessed with an endothelial tube formation assay using human umbilical vascular endothelial cells (HUVEC). RUPP increased AP compared to Sham (123±2 vs 105±3 mmHg; P<0.05) and AP was not altered in the RUPP+ED (n=8; 117±4mmHg) or Sham+ED (n = 7; 98±3 mmHg). RUPP decreased free VEGF compared to Sham and Sham+ED rats (465.4 vs 842.9 vs 809.8 pg/ml; P<0.05) and this was restored in the RUPP+ED (809.8 pg/ml). Exercise increased (P<0.05) free VEGF in the RUPP but not the Sham rats. HUVEC tube length was decreased in RUPP compared to Sham and Sham+ED rats (61194 vs 74865 vs 79318 μm; P<0.05) and this reduction was attenuated in the RUPP+ED (71552 μm) rats. RUPP, RUPP+ED and Sham+ED had decreased fetal weight compared to Sham (2.3 vs 2.0 vs. 2.2 vs 2.2 vs2.6g; P<0.05). In contrast to our previous report that exercise before and during pregnancy increased free plasma VEGF in normal pregnant and sham rats and decreased AP and restored angiogenic balance in RUPP rats, the present data demonstrate that an exercise regimen started at pregnancy in rats that are exercise naïve at conception has both beneficial and detrimental effects on maternal-fetal outcomes in a gestation complicated by placental-ischemia.
Abstract 271: Does Obesity Alter the Normal Cardiovascular and Renal Adaptations of Pregnancy?
Authors: Cai Xiaochu , Kett Michelle M
Published: March 2022

Obesity is associated with significant complications during pregnancy, including hypertensive disorders of pregnancy, and increased risk of cardiovascular and renal disease later in life. Surprisingly, we know little about the impact of obesity on the cardiovascular and renal adaptations that occur during a normal pregnancy. The aim of present study was to determine the adaptations that occur in mean arterial pressure (MAP), heart rate (HR) and renal function across pregnancy in a mouse model of diet-induced obesity.

At 4wks of age, female C57BL6J mice were place on a control (Cont; 16% fat, 3.85 kCal/g; n=8) or high fat (HF; 43% fat, 4.54 kCal/g; n=7) diet for 10 weeks and glucose tolerance tests (GTT) performed. Baseline MAP and HR (radio-telemetry), and 24hr urinary albumin excretion (n=3-5/gp) were measured. Mice were then mated, and MAP and HR measured throughout gestation with 24hr albumin excretion reassessed at gestational day (GA) 13.

After 10 wks of the diet, body weight of HF mice was 40% greater than Cont mice (33.2±1.8 vs 23.6±0.6g; P<0.001). Both groups of mice had similar fasting blood glucose. The obese mice were, however, glucose intolerant compared to Cont mice (725±60 vs 467±64, Area Under the Curve; P<0.05), had elevated basal MAP (105.2±2.1 vs 99.3±1.6mmHg; P<0.05), HR (556±13 vs 492±17bpm; P<0.05) and were albuminuric (4.53±0.62 vs 2.42±0.42μg/24h; P<0.05). Following mating, MAP (Pdiet<0.01) and HR (Pdiet<0.05) of obese mice remained elevated over Cont mice throughout pregnancy. However the adaptations of MAP and HR in obese mice over pregnancy, including the dip in MAP at GA9, were consistent in timing and magnitude with Cont mice. Pregnancy led to a significant increase in 24hr albumin excretion at GA13 (Ppreg<0.001), however this effect was exacerbated in obese mice (9.95±0.88 vs 4.03±1.1μg/24h; PpregXdiet <0.01).

Obesity results in higher MAP and HR before pregnancy and both remain elevated throughout pregnancy without altering the normal adaptations of MAP and HR during pregnancy. Importantly, pregnancy exacerbates albuminuria in obese females suggesting that obese pregnancy might increase the risk of renal complications during pregnancy and lead to renal disease in the long-term.
Abstract 272: Reduced Uterine Perfusion Pressure Stimulated T-helper 17 Cells Cause Hypertension during Pregnancy
Authors: Cornelius Denise C , Wallace Kedra , Moseley Janae , LaMarca Babbette
Published: March 2022

Preeclampsia (PE), new onset hypertension with proteinuria during pregnancy, is associated with chronic inflammation and an imbalance among T-helper cell subtypes. Adoptive transfer of all CD4+ T cells from Reduced Uterine Perfusion Pressure (RUPP) rats into Normal Pregnant (NP) rats caused elevations in blood pressure and Angiotensin II type I receptor autoantibody (AT1-AA) and inflammatory cytokines. We have shown that chronic IL-17 infusion increases blood pressure and AT1-AA during pregnancy. Furthermore, we have shown that blockade of IL-17 decreases blood pressure and AT1-AA in the RUPP rat model of PE. The objective of this study was to determine a role for the elevated autoimmune associated T-helper 17 (T
H
17) cells from RUPP rats to cause hypertension and chronic inflammation during pregnancy. CD4
+
CD25
-
T

cells were isolated from RUPP rat spleens using biotinylated antibody. Isolated cells were then stimulated on CD3 and CD28 magnetic beads and cultured in T-helper media to differentiate cells into T
H
17 cells. Differentiation of isolated cells into T
H
17 cells was verified via flow cytometry. On day 12 of gestation, 1x10
6
T
H
17 cells from RUPP rats were adoptively transferred into NP rats. Carotid catheters were inserted on gestation day18, and on day 19 blood pressure (MAP) was recorded, serum and plasma were collected. One-way ANOVA was used for statistical analysis. MAP increased from 99 ±2 mmHg in NP (n=8), to 128±4 mmHg in RUPP (n=9, P<0.0001). Likewise, adoptive transfer of RUPP T
H
17 cell into NP (NP + RUPP T
H
17) significantly increased MAP to 111±3 mmHg (n=11, P<0.001 compared to NP). AT1-AA significantly increased from 0.1±0.2 beats/min in NP to 15.6±0.7 beats/min in NP + RUPP T
H
17 rats (p<0.01). Plasma inflammatory cytokines were assessed via ELISA. IL-6 increased from 27.5±7.3 pg/mL in NP to 60.3±9.5 pg/mL in NP + RUPP T
H
17 rats (p<0.05); TNF-α increased from 11.4±9 pg/mL in NP to 19.9±6 pg/mL in NP + RUPP T
H
17; IL-17increased from 0.49±0.32 pg/ml in NP to 5.46±5 pg/mL in NP +RUPP T
H
17. In conclusion, RUPP T
H
17 cells increased blood pressure, AT1-AA, and inflammatory cytokines when transferred to NP rats, indicating a role for autoimmune associated T
H
17 cells, to cause much of the pathophysiology associated with PE.
Abstract 278: Enhanced Vasorelaxation Response of Resistance Arteries From Obese Mc4r Deficient Pregnant Rats
Authors: Spradley Frank T , Palei Ana C , Granger Joey P
Published: March 2022

While obesity is a major risk factor for preeclampsia, the mechanisms linking obesity and hypertension during preeclampsia are unclear. Hypertension in preeclampsia is associated with placental ischemia-induced release of factors such as anti-angiogenic sFlt-1 into the maternal circulation, which antagonizes vascular endothelial growth factor (VEGF) and endothelial function. The purpose of this study was to determine the effect of obesity on angiogenic factors and endothelial function in obese melanocortin 4 receptor (MC4R) knockout rats (MC4R+/-, n=4-9) and lean control MC4R+/+ rats (n=4-8). Pre-pregnancy body weight (298±6 vs. 266±3g) and total body fat mass (EchoMRI: 56±3 vs. 44±1g) was greater (P<0.05) in MC4R+/- vs. MC4R+/+ at 15 weeks old when timed-pregnant rats were generated. At gestational day (GD) 18, body weight (362±6 vs. 336±5g) and total body fat mass (62±6 vs. 45±5g) were greater (P<0.05) in MC4R+/- and MC4R+/+ pregnant rats, respectively. Mean arterial blood pressure (indwelling carotid catheter) was similar in MC4R+/- (113±4mmHg) and MC4R+/+ (109±4mmHg) rats. Plasma sFlt-1 was not significantly elevated in MC4R+/- vs. MC4R+/+ (228±86 vs. 141±53 pg/mL). Interestingly, VEGF was greater in the obese pregnant rats (4091±336 vs. 2854±475pg/mL, P<0.05). Wire myography demonstrated in third-order mesenteric arteries that sensitivity (logEC50) to acetylcholine (endothelial dependent)-induced vasorelaxation (-11.4±0.7 vs. -6.9±0.7M, P<0.05) and sodium nitroprusside (exogenous nitric oxide donor) (-8.3±0.1 vs. -7.5±0.2M, P<0.05) was greater in obese MC4R+/- vs. lean MC4R+/+ pregnant rats. These data indicate that during normal pregnancy, obesity is associated with greater circulating VEGF levels and nitric oxide-dependent vasorelaxation in resistance arteries. Because soluble factors from the ischemic placenta target and reduce nitric oxide bioavailability, we propose that the hypertensive and endothelial dysfunction responses to reductions in uterine perfusion may be exaggerated in obese pregnant animals.
Abstract 281: HO-1 Induction Increases ßENaC in Ischemic Placentas and Cultured Cytotrophoblasts
Authors: Warrington Junie P , George Eric M , Stec David E , Ryan Michael J , Granger Joey P , Drummond Heather A
Published: March 2022

Preeclampsia is characterized by abnormal placentation that is thought to arise from inadequate cytotrophoblast migration and invasion of the spiral arteries. The resulting reduced perfusion of the utero-placental unit promotes the development of hypertension in preeclampsia. Beta Epithelial Na+ Channel (βENaC) protein, a mediator of cytotrophoblast migration in vitro, is decreased in placentas of preeclamptic patients. We have recently reported that hemeoxygenase-1 (HO-1) and its by-products reduce blood pressure in placental ischemic rats. Since HO and its byproducts can regulate ENaC expression, including βENaC, are important mediators of migration, the goal of this study was to test the hypothesis that HO-1 induction 1) stimulates βENaC expression and migration in cultured cytotrophoblasts and 2) restores βENaC expression in ischemic placentas. Cobalt protoporhyrin (CoPP, 1 - 10 μM, 48 hrs) stimulated HO-1 induction in cultured cytotrophoblasts (BEWO cells), upregulated βENaC expression, and increased spontaneous migration by 39.2 ± 6.0% (n=7, p<0.001). To determine if HO-1 induction increased placental βENaC expression and prevented hypertension, CoPP was injected subcutaneously (5mg/kg) on gestational day 14 in timed pregnant Sprague-Dawley rats with placental ischemia. MAP was increased on gestational day 19 in placental ischemic rats (141 ± 4 vs.101 ± 1 mmHg, p<0.001; n=4 per group). While HO-1 induction did not affect MAP in normal pregnant rats (101 ± 5 mmHg), MAP was significantly attenuated (p<0.01) in placental ischemic rats treated with CoPP (117 ± 1 mmHg). Western blot analysis revealed a 62 ± 22% decrease in βENaC expression in ischemic placentas, which was restored by HO-1 induction (n= 4, p<0.05). These findings suggest that HO-1 induction stimulates βENaC expression in cultured cytotrophoblasts and ischemic placentas. Future studies will determine if the beneficial effects of HO-1 occur through βENaC-mediated cytotrophoblast migration and spiral artery remodeling in ischemic placentas. Targeting the HO-1 pathway presents a potential therapeutic approach for improving placentation in preeclamptic women.
Abstract 282: Aromatic Residue-based Tetrapeptide Fqqi Motif Mediates Internalization and Trafficking of Guanylyl-cyclase/natriuretic Peptide Receptor-a in Mouse Mesangial Cells
Authors: Mani Indra , Garg Renu , Pandey Kailash N
Published: March 2022

Atrial natriuretic peptide (ANP) is a cardiac hormone that modulates blood pressure and blood volume by activation of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and subsequent generation of intracellular second messenger cGMP. The aim of the present study was to examine the role of small sequence motifs in the internalization and trafficking pathway of NPRA by immunofluorescence confocal microscopy. A chimeric protein of enhanced green fluorescence protein (eGFP) and NPRA (eGFP-NPRA) was used to study the dynamics of NPRA trafficking in the intact mouse mesangial cells (MMCs). We have identified a FQQI (Phe
790
, Gln
791
, Gln
792
and Ile
793
) motif in the carboxyl-terminal domain of NPRA sequence and mutated with alanine residues (FQQI/AQQA) using QuickChange II site-directed mutagenesis kit. Cells were transiently transfected with mutated and wild-type constructs and cultured at 37
0
C in an atmosphere of 5% CO
2
and 95% O
2
. In the ANP-treated cells, colocalization of wild-type eGFP-NPRA with pan-Cadherin, a plasma membrane marker, indicated that internalization of receptor accounted for 59.2%±3.4, 70.0%±4.3, 71.8%±4.3, and 77.4%±3.2 at 5, 10, 15, and 30 min, respectively of total fluorescence intensity compared with controls. Colocalization of eGFP-NPRA with early endosome antigen-1 (EEA-1) marker was maximum at 5 min (64.5%±2.3), and gradually decreased at 30 min (24.4%±3.8). However, the mutation of FQQI/AQQA decreased

the receptor internalization by 45% (
P<
0.001) and the receptor colocalization in to endosome by 58% (
P
<0.0001), respectively, compared with wild type receptor. After ANP treatment, immunofluorescence analysis showed a marked increase in the accumulation of intracellular cGMP (29-fold) concurrent with receptor internalization compared with controls. The results indicate that ligand-receptor complexes continue to traffic and produce cGMP even after receptor sequestration into endosomes. Our, data demonstrate that FQQI motif plays essential role in the internalization of NPRA, which regulates hypertension and cardiovascular homeostasis.
Abstract 298: Chronic Environmental Enrichment Improves Aging-related Endothelial Dysfunction in Rats: Role of Oxidative Stress
Authors: Idris-Khodja Noureddine , Pereira de Vasconcelos Anne , Auger Cyril , Barbelivien Alexandra , Majchrzak Monique , Cassel Jean-Christophe , Schini-Kerth Valérie B
Published: March 2022

Aging is associated with an endothelial dysfunction, which promotes cardiovascular diseases. Physical exercise has beneficial effects on the endothelial function. However, the effect of an enriched environment (EE) promoting physical exercise, on the endothelial function has not been explored during aging. We investigated the endothelial dysfunction in Long-Evans female rats housed in standard conditions (SD, 2 rats per cage) until the age of 18 months. Then, half the rats were transferred to EE (12 rats in one large cage containing various objects, which were changed 5 times per week) for 6 months. A group of 2-month old rats served as young controls. Systolic (SBP), diastolic (DBP) blood pressure and heart rate (HR) were determined by plethysmography. Vascular reactivity was assessed in mesenteric arteries using organ chambers, vascular oxidative stress by dihydroethidine staining, and the proteins expression by immunofluorescence. Aging under SD was associated with increased SBP (by 40 mmHg,
P
<0.001), DBP (by 12 mmHg,
P
<0.001) and HR (by 110 bpm,
P
<0.001) compared to young rats. It was also associated with blunted acetylcholine (ACh)-induced endothelium-dependent nitric oxide (NO)-mediated relaxations (EC50 1.08 10
-8
M vs. 3.08 10
-8
M,
P
<0.05) and endothelium-derived hyperpolarization (EDH)-mediated relaxation (E
max
89.5±11.8% vs. 0.28±0.18%,
P
<0.001), and the induction of endothelium-dependent contractile responses to ACh (E
max
0.004±0.008 g vs. 0.71±0.07 g,
P
<0.001) in mesenteric artery rings. EE reduced SBP and DBP by 18 and 10 mmHg, respectively (
P
<0.05), HR by 30 bpm (
P
>0.05), and improved NO-mediated responses to a similar level as in young rats (
P
<0.001) without affecting the blunted EDH-mediated relaxation and the increased endothelium-dependent contractile responses in old rats. An excessive oxidative stress and the upregulation of endothelial NO synthase (eNOS), nitrotyrosine and arginase I were observed in the aorta of SD old rats compared to young rats (
P
<0.001). EE improved all these effects (
P
<0.05). In conclusion, EE retarded the aging-related endothelial dysfunction mostly by improving the NO-mediated relaxation. This effect involves normalization of vascular oxidative stress and possibly also eNOS uncoupling.
Abstract 299: Remodeling of Endothelial Function in Atherosclerotic Mice Overexpressing Endothelin-1 Restricted to Endothelium
Authors: Idris-Khodja Noureddine , Mian Muhammad Oneeb Rehman , Li Melissa W , Leibowitz Avshalom , Paradis Pierre , Rautureau Yohann , Schiffrin Ernesto L
Published: March 2022

Background:
In human atherosclerosis , which is associated with elevated plasma and coronary endothelin (ET)-1 levels, ET type A receptor antagonists improved coronary endothelial function. Mice overexpressing ET-1 specifically in the endothelium (eET-1) crossed with apolipoprotein E knockout mice (Apoe-/-) exhibited exaggerated high-fat diet (HFD)-induced atherosclerosis. Since endothelial dysfunction often precedes development of atherosclerosis, we investigated whether endothelium-specific ET-1 overexpression causes endothelial dysfunction in Apoe-/- mice.

Methods:
Eight-week male old eET-1, Apoe-/-, eET-1/Apoe-/- and wild-type (WT) mice were fed a regular diet or HFD for 8 weeks. Endothelial function was assessed in mesenteric arteries by pressurized myography.

Results:
In HFD-fed mice, acetylcholine-induced endothelium-dependent relaxation (EDR) was reduced in Apoe-/- and eET-1 compared to WT (Emax: 29.2±2.9% and 51.8±7.9%, respectively, vs. 88.0±2.6%, P<0.05). Surprisingly, EDR was not impaired in eET-1/Apoe-/- (70.9±9.8%) compared to WT. Endothelium-independent relaxation to the nitric oxide (NO) donor sodium nitroprusside and contractile responses to norepinephrine were unaffected. Similar results were observed in regular diet-fed mice. In the presence of inhibitors of either NO synthase (NOS)-mediated relaxation, Nω-nitro-L-arginine methyl ester, or endothelium-dependent hyperpolarization (EDH)-mediated relaxation, apamin plus Tram34, EDR was blunted in WT (Emax: 2.8±0.4% and 35.7±8.4 respectively, P<0.01), whereas relaxation was weakly reduced or unaffected in eET-1/Apoe-/- (Emax: 68.6±9.9, P<0.05 and 80.6±8.0, respectively). However, the concomitant inhibition of NOS- and EDH-mediated relaxation strongly reduced EDR in eET-1/Apoe-/- (Emax: 43.0±8.2%, P<0.05).

Conclusions:
These results show an interdependence of NOS and EDH pathways in EDR in WT mice. ET-1 overexpression induced development of compensatory mechanisms in pre-atherosclerotic arteries of Apoe-/- mice, which permits either NOS or EDH pathway to mediate independently EDR. Investigation of mechanisms involved in the remodeling of EDR in eET-1/Apoe-/- mice will allow a better understanding of the ET-1 role in atherosclerosis.
Abstract 303: A Novel Adipocytokine, Omentin Prevents Vascular Smooth Muscle Cell Migration by Inhibiting O
2
-/p38/HSP27 Pathway
Authors: Kazama Kyosuke , Okada Muneyoshi , Yamawaki Hideyuki
Published: March 2022

(Background)
Omentin, a novel adipocytokine, is mainly expressed in the visceral rather than subcutaneous adipose tissue. Secretion and plasma concentration of omentin decrease in the obese subjects. We previously demonstrated that omentin is anti-inflammatory in vascular smooth muscle cells (SMCs). While vascular remodeling via proliferation, migration and apoptosis of SMCs is also important for hypertension development, it remains to be clarified whether omentin affects those processes.

(Methods and Results)
Cultured rat mesenteric arterial SMCs were stimulated with platelet-derived growth factor (PDGF)-BB (10 ng/ml) in the absence or presence of omentin (300 ng/ml). Omentin pretreatment (2 h) inhibited PDGF (6 h)-induced SMCs migration (30 % inhibition, n=11, P<0.05) as determined by Boyden camber assay. Omentin pretreatment (2 h) inhibited NADPH oxidase -derived O2- generation as determined by dihydroethidium staining and lucigenin assay (30 % inhibition, n=5, P<0.05) as well as phosphorylation of p38 (20 % inhibition, n=19-23, P<0.01) and heat shock protein (HSP) 27 (20 % inhibition, n=14-17, P<0.01) as determined by Western blotting. Omentin pretreatment (2 h) inhibited PDGF (6 h)-induced lamellipodia formation as determined by rhodamine-phalloidin staining. In ex vivo, omentin pretreatment (6 h) also inhibited fetal bovine serum (FBS) (10%)-induced SMCs out-growth from rat isolated mesenteric artery.

(Conclusion)
We for the first time demonstrate that omentin prevented SMCs migration by preventing the activation of ROS-dependent p38/HSP27 signals. It is suggested that omentin is anti-hypertensive ‘good adipocytokine’.
Abstract 306: Lipoxin A4 Mediates Aortic Contraction and Endothelial Dysfunction via Reactive Oxygen Species
Authors: Wenceslau Camilla F , McCarthy Cameron G , Goulopoulou Styliani , Szasz Theodora , Webb R. Clinton
Published: March 2022

Lipoxin A4 (LX4) is a biologically active product generated from arachidonic acid by lipoxygenase action and its formation is not inhibited by aspirin. In fact, aspirin triggers the production of lipoxins through acetylation of cyclooxygenase 2 (COX-2) that metabolizes arachidonic acid to 15(R)-hydroxyeicosatetraenoic acid. This metabolite is then converted, via lipoxygenase, to lipoxin, also known as “aspirin-triggered lipoxin”. LX4 has both anti-inflammatory and proinflammatory actions that are related with anaphylaxis, reocclusion and restenosis after coronary angioplasty in patients treated with aspirin. The formation of LX4 within the vascular lumen and wall during inflammation, e.g. after aspirin treatment, places this lipid in a strategically advantageous site for modulation of vascular function. However, little is known of the actions of LX4 on the vasculature. We hypothesized that LX4 would elicit contractile responses and will induce endothelial dysfunction in rat aorta. We used aorta from 12-week old male Wistar rats to assess vascular function on the pin myograph and reactive oxygen species (ROS) production via dihydroethidium fluorescence. LX4 (1-300 nM) induced concentration-dependent contractions and indomethacin, a COX inhibitor (INDO, 10 μM), did not affect this result (E
max
, LX4: 30±6
vs.
INDO: 20±1 % of KCl, p>0.05). Also, concentration-response curves to acetylcholine (ACh, 0.1 nM-30 μM) and phenylephrine (PE, 1 nM-30 μM) were performed in the absence (vehicle) or presence of LX4 (1 nM). LX4 increased aortic sensitivity to PE (pD
2
, vehicle: 7.37±0.05
vs.
LX4: 7.64±0.07, p<0.05) and decreased ACh-induced relaxation (E
max
, vehicle: 86±3
vs.
LX4: 67±3 % of PE, p<0.05). Finally, incubation of aortic segments with LX4 (1 nM) for 15 min significantly increased dihydroethidium fluorescence (0.33±0.01 fold compared to vehicle, p<0.05), suggesting that LX4 may induce oxidative stress. In conclusion, these results show that LX4 has a functional role in aorta and may contribute to vascular dysfunction via increases in ROS. This lipid may contribute to further vascular damage in conditions where its production is exacerbated, such as in atherosclerosis or angioplasty-associated complications treated with aspirin.
Abstract 319: Vascular Oxidative Stress Promotes Aortic Stiffening
Authors: Wu Jing , Chen Wei , Harrison David G
Published: March 2022

Clinical studies have shown a relationship between markers of oxidative stress and parameters of arterial stiffness. We have previously found that vascular reactive oxygen species (ROS) contribute to aortic hypertrophy in hypertension. In the present study, we hypothesize that vascular oxidative stress induces aortic stiffening by causing oxidative injury to the vessels. To test this hypothesis, we first studied isolated aortas from tg
sm/p22phox
mice, which have a constitutive increase in vascular ROS production. Six month old tg
sm/p22phox
mice were normotensive at baseline (118±6 systolic, 89±5 diastolic, mean±S.E.M. mmHg), but had increased aortic stiffness as indicated by the leftward shift on stress-strain curves (p<0.05 vs. wild type, one way ANOVA). While treatment with angiotensin II increased aortic stiffness in wild-type mice, it did not further increase stiffness in the tg
sm/p22phox
mice. In order to understand the nature of ROS involved in aortic stiffening, we deleted extracellular superoxide dismutase (ecSOD) in vascular smooth muscle using cre-loxp technology. Deletion of ecSOD would increase O
2
·-
, but not H
2
O
2
. Surprisingly, we found no difference in aortic stiffness or blood pressure in these mice compared to wild-type mice, either at baseline or in response to angiotensin II. These data indicate that O
2
·-
is unlikely involved in increasing aortic stiffness. It is therefore likely that other ROS, such as H
2
O
2
, modulate this response. In this regard, we have performed additional studies in which mice were treated with salicylamine, a scavenger of isoketals, which are oxidized products of arachidonic acid. These are known to cross-link lysines and could promote cross-linking of cellular components leading to vascular stiffening. Amazingly, this drug markedly reduced blood pressure (133±8 vs. 168±9 systolic; 95±6 vs. 125±7 diastolic, salicylamine vs. angiotensin II alone, p<0.01) and aortic stiffening in response to angiotensin II. In conclusion, our data strongly suggest that vascular ROS promotes aortic stiffening by inducing oxidative injury to the vessels.
Abstract 350: Downregulation of C-terminal Src Kinase (csk) and Csk-binding Protein (cbp) is Associated With Increased Aldosterone-induced C-src Phosphorylation in Shr Vascular Smooth Muscle Cells
Authors: Callera Glaucia E , Montezano Augusto C , Bruder-Nascimento Thiago , Touyz Rhian M
Published: March 2022

c-Src phosphorylation is controlled by the recruitment of enzyme regulators, such as C-terminal Src kinase (CSK) which inhibits Src activity, and interactions with transmembrane adaptors. These complex regulatory mechanisms coordinate activity of c-Src at multiple levels. We previously showed that in aldosterone-stimulated SHR vascular smooth muscle cells (VSMCs), c-Src phosphorylation and its downstream signaling are upregulated. Here we hypothesized that mechanisms underlying vascular c-Src hyperactivation in SHR are related to dysregulated CSK and altered autophosphorylation at Tyr416 and Tyr527 in aldosterone-stimulated SHR cells. Studies were performed in cultured VSMCs from WKY and SHR. C-terminal Src kinase (Csk) cytosol/membrane translocation, Csk-binding protein (CBP), and c-Src phosphorylation were evaluated by western blot. Cholesterol-enriched fractions were obtained by sucrose-gradient centrifugation. Aldosterone (100 nM) induced Tyr527 c-Src phosphorylation (153.5 ± 13.6 %) which locks the kinase in an inactive conformation. This was blunted in SHR cells. Csk is a cytosolic kinase that catalyzes c-Src Tyr527 phosphorylation. ASN (10 uM), a Csk inhibitor, prevented the kinase translocation to the membrane and inhibited Tyr527 c-Src phosphorylation induced by aldosterone in WKY cells. Inhibition of Csk induced an increase in Tyr416 c-Src (180 ± 21%) under basal conditions. In SHR cells, Csk translocation to the membrane was reduced by aldosterone compared with WKY cells. Aldosterone induced an increase in expression (180.3 ± 39 %) and phosphorylation (169 ± 23 %) of the adaptor protein CBP in WKY, but not in SHR cells. Aldosterone stimulation increased Csk trafficking into lipid rafts/caveolae in WKY cells, without affecting the kinase content in the cholesterol-enriched fractions from SHR. Our findings demonstrate that 1) key regulators of c-Src activation by aldosterone, specifically Csk and CBP, are altered in SHR VSMCs; 2) c-Src regulation by aldosterone involves lipid rafts/caveolae. These novel findings suggest that modulation of Csk could be an important strategy to blunt c-Src-dependent aldosterone vascular effects.
Abstract 355: Mineralocorticoid Receptor Activation Alters Epoxyeicosatrienoic Acid Production In Vivo
Authors: Luo Pengcheng , Gangadhariah Mahesha , Wei Shouzuo , Capdevila Jorge , Brown Nancy J , Pozzi Ambra , Luther James M
Published: March 2022

Epoxyeicosatrienoic acids (EETs) are products of CYP450 epoxygenase and are degraded to DHETs by soluble epoxide hydrolase (sEH). Their natriuretic and vasodilatory actions oppose those of mineralocorticoids, but the effect of aldosterone on EET metabolism has not been investigated. To test the hypothesis that aldosterone impairs EET metabolism via altered production or degradation, we treated wild type C57Bl6J mice with aldosterone (50 μg/kg/d) or vehicle ± the mineralocorticoid receptor (MR) antagonist RU-28318 during high sodium intake for 2 weeks. Aldosterone decreased plasma (
Fig A
; -29%, 4.4±0.6 vs. 3.1±0.4 ng/ml, control vs. aldosterone) and pancreatic EETs (
Fig B
;-43%, 112±15 vs. 64±15 ng/mg tissue, control vs. aldosterone) but not hepatic or kidney EETs. MR antagonism prevented this effect of aldosterone on plasma EETs. The urinary DHET/EET ratio markedly increased, suggesting increased renal sEH activity. Because both aldosterone and EETs affect insulin secretion, we further assessed CYP epoxygenase expression within the pancreas. We detected
Cyp2c44
and sEH (
Ephx2)
mRNA in isolated murine islets, and immunohistochemistry demonstrated CYP2C expression in beta-cells. Aldosterone decreased protein expression of CYP2C epoxygenase within whole pancreas, and
ex vivo
aldosterone treatment decreased
Cyp2c
and increased
sEH
mRNA expression within isolated islets. These data demonstrate that aldosterone decreases CYP2C expression and EET production
in vivo
. We propose that the renin-angiotensin-aldosterone system interacts with the CYP450 epoxygenase system to alter blood pressure and glucose homeostasis via EET metabolism.
Abstract 359: Modulation of Aldosterone Synthase by Estrogens: Evidence for an Interaction of Gper-1 and Estrogen B Receptors and Relevance for the Gender Dimorphism of Blood Pressure
Authors: Caroccia Brasilina , Seccia Teresa M , Lenzini Livia , Kuppusamy Maniselvan , Gonzalez Campos Abril , Mareso Sara , Gioco Francesca , Fassina Ambrogio , Rainey William E , Rossi Gian Paolo P
Published: March 2022

Background.
Fertile women have lower blood pressure and are held to be at lower risk for cardiovascular events than age-matched man. However, whether this gender dimorphism involves a modulation of aldosterone synthesis by 17 beta-estradiol (E2) is unknown.

Aims.
i) To investigate estrogen receptor subtypes gene expression in the normal human adrenal cortex (NAC), in aldosterone producing adenoma (APA) and in a human adrenocortical carcinoma cell line (HAC15); ii) To assess the effect of E2 on aldosterone synthase (CYP11B2) gene expression and to identify the receptor subtypes involved in this effect.

Methods.
We measured the expression of alpha (ERa), beta (ERb) and of G protein-coupled receptor (GPER-1)-1 in NAC and in APA tissue, and in HAC15 cells by real time RT-PCR. After demonstration that HAC15 cells express ERa, ERb, and GPER-1 we stimulated cells with 10-7M E2 alone, or after ERb selective blockade with 10-5M tetrahydrochrysenediol (THC), ERa selective blockade with 10-5M MPP dihydrochloride (MPP), non selective ERa and ERb blockade with 10-5M ICI 182.780, or after selective GPER-1 receptor blockade with 10-5M G-15. The cells were also exposed to the GPER-1 agonist G-1, alone or in the presence of MPP, THC, or Fulvestrant, and/or G-15. Changes of expression of CYP11B2 mRNA, measured with RT RT-PCR, was the experimental endpoint.

Results.
The quantitative expression of estrogen receptor subtypes was ERb > GPER-1 >> ERα in NAC, GPER-1 > ERb> ERa in APA, and ERb>ERa=GPER-1 in HAC15 cells. E2 alone or on top of selective ERa antagonism did not alter CYP11B2 expression. By contrast, E2 significantly increased CYP11B2 expression (+500 to + 700% from baseline, p <0.001) after selective ERb antagonism, or combined ERa and ERb blockade. Likewise, G-1 markedly increased CYP11B2 gene after combined ERb blockade, an effect that was abolished by G15 co-treatment.

Conclusion.
E2 potently stimulates aldosterone synthase expression via GPER-1 subtype receptor activation when the ERb is blocked. The ERb-mediated tonic inhibition of aldosterone synthase could contribute to explaining both the lower BP and CV risk of fertile women and the increase of BP after when this tonic ERb-mediated inhibition wanes during menopause or estrogen-modulation treatment.
Abstract 361: Episodic High Blood Pressure in Childhood Predicts Adult Hypertension
Authors: Tu Wanzhu , Eckert George , Hannon Tamara , Rosenman Marc , Pratt Linda , DiMeglio Linda , Pratt J. Howard
Published: March 2022

Occasional high blood pressure (BP) readings in children are common; however, their clinical significance has not been carefully evaluated. In this study, we examined the association between childhood BP and adulthood hypertension in a prospectively followed cohort. The original cohort was established in 1986 and consisted of 1117 healthy children (528 male and 589 female), recruited from schools in Indianapolis, Indiana (mean age 12.1; SD 5.7 years). During the course of follow-up, BP was measured semiannually with a mean of 8.4 visits per child (SD 7.2). For the active subjects that were still being followed (n=263), diagnosis of hypertension was made based on JNC-7 criteria. For subjects no longer active in the study (n=854), we examined their adult medical records for physician-diagnosed hypertension. The mean age of the subjects as of 2013 was 33.4 years (SD 3.9). Among the adult subjects, there were 119 cases of hypertension (31 identified through prospective follow-up, 88 through chart review). We examined the childhood BP records of the study subjects using age, sex, and height adjusted BP percentile values; readings exceeding the 95th percentile were considered to be in a hypertensive range. In children with no BP measures exceeding the 95th percentile, the adult hypertension rate was 8.6% (82/959); in children with one BP measure in the hypertensive range, adult hypertension rate increased to 18% (20/110); in children with two or more BP measures in the hypertensive range, the adult hypertension rate was 35% (17/48). Adjusting for the effects of age, sex, and body mass index, we found that having at least one BP reading in the hypertensive range in childhood significantly increased the adult hypertension risk (adjusted odd ratio=3.1, 95%CI: 2.0-4.8). In addition, it is noteworthy that 59% (70/119) of those with hypertension as adults were either overweight or obese as children. In conclusion, episodic high BP in childhood significantly predicts adult hypertension risk. Occasional high BP readings should not be dismissed and modifiable risk factors should be addressed.
Abstract 365: Microalbuminuria is the Main Associated Factor with Lack of Blood Pressure Control in Patients taking 2 or 3 Antihypertensive Drugs
Authors: Oliveras Anna , Armario Pedro , Lucas Silvia , de la Sierra Alejandro
Published: March 2022

Despite increased therapeutic efforts, a substantial number of hypertensives do not reach BP goal. The knowledge of the factors associated with uncontrolled BP might help to identify this cohort and perhaps differently approach their treatment.

Aim:
To assess the prevalence of BP control (defined as BP <140/90 mmHg) and its associated factors, in patients treated with 2 or 3 antihypertensive drugs.

Methods:
In this observational, cross-sectional study, 1337 consecutively attended hypertensives (59% males, aged 63) treated with 2 or 3 antihypertensive drugs were evaluated in 36 referral centers in Spain. Office BP was measured and clinical and laboratory data were recorded. Microalbuminuria was defined as urinary albumin/creatinine ratio ≥22 (men) or ≥31 (women) and <300 mg/g in all cases. Established CV or renal disease was defined by the diagnosis of heart disease, cerebrovascular disease, peripheral vascular disease, advanced retinopathy and/or nephropathy [if serum creatinine >1.5 mg/dl (men) or >1.4 (women) and/or estimated glomerular filtration rate < 60ml/min/1.73m
2
and/or macroalbuminuria (≥ 300 mg/g)].

Results:
Seven-hundred sixty-seven patients (57.4%) had BP at goal (<140/90 mmHg). The prevalence of controlled BP according to the presence (yes vs. not) of risk factors was: diabetes: 52% vs. 60% (p=0.005); microalbuminuria: 49% vs 61% (p<0.0001). P=NS for the remaining: male sex: 58% vs. 56%; age >65yr: 55% vs. 46%; smoker: 57% vs. 58%; family CVD history: 57% vs. 60%; dyslipidemia: 56% vs. 61%; duration of hypertension >10yr: 56% vs. 59%; abdominal obesity: 55% vs. 60%; established CV or renal disease: 57% vs. 58%.

A logistic regression analysis was performed, with controlled/uncontrolled BP as the dependent variable and the following independent variables (those with p<0.2 in bivariate analyses): age, diabetes, dyslipidemia, abdominal obesity, microalbuminuria and duration of hypertension >10yr. The microalbuminuria was the only variable that remained in the model: MOR (95%CI): 0.626 (0.483-0.810); p<0.05.

Conclusions:
In hypertensives treated with 2 or 3 antihypertensive drugs, the microalbuminuria was the only factor independently associated to the lack of BP control. The prevalence of controlled BP in this cohort was 57.4%.
Abstract 38: Exosomes Secreted From Human Renal Proximal Tubule Cells Can Transfer Their Contents to Proximal Tubule, Distal Tubule and Collecting Duct Cells
Authors: Gildea John J , Reyes Camellia M , Bigler Wang Dora , Pettigrew Abigail C , Carlson Julia M , Felder Robin A
Published: March 2022

Exosomes are 50-90 nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have previously found that urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. We also found that urinary exosomes contain dopamine 1 receptor and G protein-coupled receptor kinase type 4. We have also shown that exosomes can transfer between cultured human renal proximal tubule cells (RPTC). Here, we further explored RPTC-to-RPTC exosomal transfer using 5 RPTC lines containing exosomes that were fluorescently labelled with either exosomal-specific marker CD63-EGFP or CD9-mCherry. Transfer between RPTCs was confirmed by co-culturing CD63-EGFP and CD9-mCherry stable RPTC clones and performing live confocal microscopy. Dual color time-lapse videos reveal unique cellular secretion and uptake patterns for exosomes and eventual accumulation into the multi-vesicular body.

We hypothesized that proximal tubule-derived exosomes could also be taken up by the distal tubule (DT). Five DT and 3 collecting duct (CD) cell lines were isolated and immortalized by infecting with hTert-containing lentiviral particles. DT cells were isolated from human kidney by collagenase digestion and biotinylated lectin or immuno-magnetic affinity separation (using dolichos biflorus agglutinin (DBA) or anti-Tamm-Horsfall protein antibody). CD cells were similarly isolated with anti-L1CAM antibody. Using sterile-filtered cell culture supernatant from 3 different CD9-mCherry RPTC lines, all 5 DT cell lines showed exosome transfer at 24 hours as measured by microplate fluorometry (10,406 ± 1132 SEM RFU above background, p<0.0001, n=30). Similarly, all 3 CD cell lines showed significant exosome transfer (8773 ± 1558 SEM RFU, p<0.0001, n=18). CD63-EGFP exosomes from 2RPTC lines also transferred to all of the DT cell lines (1713 ± 1131 RFU above background, p<0.0001, n=30) and CD cell lines (1007 ± 315 RFU, p<0.01, n=18).

In conclusion, intra-nephron cellular exosomal transfer is demonstrated in RPTCs and between RPTCs and distal tubule and collecting duct cells. This proximal to distal nephron transfer may represent a new trans-cellular communication within the human kidney.