bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P104: Renin Angiotensin System (RAS) Modulation in Hypertension Program by Maternal Intrauterine Malnutrition
Authors: Banti Rafael S , Yokota Rodrigo , Aragão Danielle S , Souza Adriana , Pedroso Amanda , Ronchi Fernanda A , Ribeiro Eliane B , Casarini Dulce E , Andrade Maria Claudina C
Published: July 2021

Intrauterine malnutrition (IM) during the early stages of development can alter the function of organs and tissues and can predict a lifetime of increased risk for adverse health outcomes, such as diabetes and hypertension. The kidney plays a key role in the development of hypertension programmed by IM, with the participation of the RAS. Our objectives were to study ACE activity and angiotensin peptides levels in tissues. Pregnants Wistar rats were separated into two groups: control group (C), fed ad libitum, and malnourished group (D) submitted to food restriction (diet 50% of the amount of feed consumed by the group C). After birth the offspring were kept as experimental groups C and D, respectively. At 4 months of age, the animals were sacrificed, heart and kidney tissues were collected to quantify angiotensin peptides and ACE activity. The offspring born with low birth weight. Kidney ACE activity was higher in group D compared to group C (299 ±86.7 vs. 253.4 ±84.82 mU/mg, p<0.05), differing from Heart (D versus C: 0.15 ± 0.08 vs. 0.24 ±0.09 mU/mg). Group D presented high blood pressure values compared to group C (140.6 ±2.8 vs. 124,3±2.6 mmHg). Kidney and heart Ang II levels were increased in group D being significant when compared to group C (238.26 ±25.1 vs. 161.85 ±45.6 pmol/g and 397.89±74.9 vs. 223.33±48.7 pmol/g, p<0.05, respectively). The same was observed for Ang I. The vasodilator peptide Ang1-7 levels in group D from kidney and heart were lower in comparison with group C, thus emphasizing an enabling environment for hypertension (220.74 ± 48.74 vs. 288.09 ± 47 pmol/g and 152.1±41.2 pmol/g vs. 228.93±41.2 pmol/g, p<0.05, respectively). Our results indicate that perturbed maternal nutritional status alters tissue RAS resulting in higher blood pressure in the offspring, demonstrated by increased renal ACE activity and Ang II levels, with reduced Ang 1-7. The increase of Ang I and II in the heart, despite low ACE activity in this tissue suggests the activation of RAS alternative pathways. This study describes for the first time that low levels of Ang 1-7 contributed to the early development of hypertension.
Abstract P112: Mechanism of the Blood Pressure Response to a Novel Vascular Disrupting Combretastatin A1-Diphosphate (OXi4503)
Authors: Shahrivari Mahan , Wise Elizabeth , Pettiford Leslie C. , Chaplin David J. , Pepine Carl J. , Cogle Christopher R.
Published: July 2021

Background:
Hypertension has been observed in cancer clinical trials of vascular disrupting agents (VDAs) but the mechanism is unknown; hence, prevention and management are difficult. Clearly, an improved understanding of the pathophysiology of blood pressure (BP) changes with VDAs would improve the standard of care for this novel class of anti-cancer agents. Accordingly, we hypothesized that VDAs may influence BP via activation of the angiotensin-renin system.

Methods:
Escalating doses of combretastatin A1-diphosphate (OXi4503) were given intravenously over 10 minutes on days 1,8,15 and 22 in 28-day cycles in a Phase IA trial of acute myeloid leukemia (AML) patients (NCT01085656). BP and plasma levels of angiotensin II (angII) were measured prior to and 0.5, 1, 2, 3, 4, 5, 6 and 24 hrs after infusion. If BP was ≥120/80 at baseline, amlodipine was administrated 60 minutes before OXi4503.

Results:
A total of 16 patients were treated: median age 63.5 years (range, 24-79); 12 were men. Two subjects received 2.5 mg/m
2
, two-3.75 mg/m
2
, nine-5 mg/m
2
and three-6.25 mg/m
2
. Most (9/16) had a history of HTN. SBP rose in all patients, reaching 140-159 mmHg in 5/16 (31.2%) and ≥160 mmHg in 3/16 (18.7%). In 11 patients pretreated with amlodipine, 5/16 (45.4%) developed SBP ≥140 mmHg. Prior HTN history and increasing age were predictors of the SBP response. In those with a HTN history, peak SBP was 151.717.2 (mean±SD) versus those without a HTN history (126.711.4, P = 0.003). At one hr after OXi4503 infusion, SBP correlated with the level of angII (Spearman: 0.53, CI95% (0.04-0.81) P= 0.03). Although there was no significant association between OXi4503 dose and percent maximal increase in SBP or level of angII, compare with baseline, there was a significant correlation between the dose and level of angll 0.5 hours after infusion (Spearman: 0.54, CI95% (0.07-0.82), P=0.02). In all patients, SBP returned to baseline by 24 hrs.

Conclusion:
The VDA OXi4503 increased BP and this may be mediated, in part, by angII signaling. Our findings suggest, for the first time, that selective angII receptor blockade is worth exploration for patients receiving VDAs.
Abstract P114: Mas Receptor Deficiency Increases Diastolic Blood Pressure and Reduces Cardiac Function in Obese Female Mice
Authors: Wang Yu , Shoemaker Robin , Thatcher Sean , Cassis Lisa
Published: July 2021

Objective:
We recently demonstrated that protection of female mice from obesity-induced hypertension was associated with increased plasma concentrations of angiotensin-(1-7) (Ang-(1-7)). Ang-(1-7) is a ligand for Mas receptors (MasR), where the peptide has been reported to promote endothelial release of nitric oxide and reduce blood pressure. In this study, we hypothesized that MasR deficiency will abolish protection of female high fat (HF)-fed mice from the development of obesity-induced hypertension.

Methods and Results:
Female (8 weeks, C57BL/6) wild type (MasR+/+) and whole body MasR deficient mice (MasR-/-) were fed a HF diet (60% Kcal as fat) for 16 weeks. MasR deficiency had no effect on the development of obesity or glucose intolerance in HF-fed female mice. At week 16 of HF feeding, blood pressure was quantified by radiotelemetry. Deficiency of MasR resulted in a significant decrease in pulse pressures of HF-fed females (24hr average: MasR+/+, 37.7 ± 1.5 mmHg; MasR-/-, 33.1 ± 1.0 mmHg; P<0.05). Diastolic blood pressures (DBP) of HF-fed female MasR-/- mice were modestly elevated during the night cycle compared to controls (DBP night: MasR+/+, 91.5 ± 2.0 mmHg; MasR-/-, 96.6 ± 1.3 mmHg; P=0.06). However, systolic blood pressure, mean arterial pressure and heart rate were not significantly different between genotypes. Assessment of left ventricular function by ultrasound demonstrated significant reductions in stroke volume (MasR+/+, 44.3 ± 1.6 μl; MasR-/-, 36.3 ± 2.1 μl), ejection fraction (MasR+/+, 57.1 ± 1.2; MasR-/-, 50.1 ± 2.7%) and fractional shortening (MasR+/+, 29.7 ± 0.8; MasR-/-, 25.3 ± 1.7%) in HF-fed MasR-/- females compared to controls.

Conclusion:
These results demonstrate that MasR deficiency promotes elevated diastolic blood pressures and reduced cardiac function in obese female mice, suggesting that the Ang-(1-7)/MasR axis protects females from obesity-hypertension. Moreover, these results suggest that MasR agonists may be effective therapies for obesity-associated cardiovascular conditions.
Abstract P135: Deletion of the Mas Receptor Aggravates Vascular Dysfunction and the Development of Atherosclerosis Through a NO-dependent Mechanism in Apolipoprotein E-deficient Mice
Authors: Yang Guang , Grave Katharina , Thieme Manuel , Hendgen-Cotta Ulrike , Rump Lars C , Stegbauer Johannes
Published: July 2021

Recently, we have shown that chronic Ang-(1-7) treatment acting through the Mas receptor improves vascular dysfunction in atherosclerotic apolipoprotein E-deficient (apoE-KO) mice by increasing NO bioavailability. To test whether deletion of the Mas receptor aggravates atherosclerosis and to examine the underlying mechanism, we generated apoE/Mas-KO mice.

12 weeks old ApoE-KO and apoE/Mas-KO mice fed on a lipid-rich Western diet were treated via osmotic minipumps with either saline or Ang-(1-7) (82 μg/kg/h) for 6 weeks. Aortae were stained with red oil and quantified for atherosclerosis. To examine whether Ang-(1-7) modulates the development of atherosclerosis through a NO dependent mechanism, 8 weeks old apoE-KO mice treated with L-NAME (20mg/kg/d) were infused either with Ang-(1-7) or saline for 6 weeks. Tissue nitrite, a marker for NO generation was measured by HPLC.

Endothelial dependent vasodilation and atherosclerosis was significantly impaired in apoE/Mas-KO mice compared to apoE-KO (relative lesion area of the aortic arch: 38.7±3.0 vs. 25.4±2.0%; P<0.01; specific lesion area 11.7±0.9 vs. 8.1±1.0mm2 P<0.01). Moreover, nitrotyrosin and urinary 8-Isoprostane levels, both markers for oxidative stress were significantly increased in apoE/Mas-KO compared to apoE-KO mice. In contrast, chronic Ang-(1-7) treatment attenuated atherosclerotic lesion in apoE-KO (11.1±2.6% vs. 25.4±2.0, P<0.01 and 3.1±0.8mm2 vs. 8.1±1.0, P<0.01) but not in apoE/Mas-KO mice (38.7±3.0 vs. 38.0±14.2% and 11.7±0.9 vs. 11.4±5.0mm2). Additionally, aortic nitrite content was increased in Ang-(1-7) treated apoE-KO compared to untreated apoE-KO mice (180±31 vs. 311±47μM/g, P<0.05). L-NAME treatment increased blood pressure (BP) and reduced aortic nitrite content significantly compared to sham-treated apoE-KO mice. However, Ang-(1-7) treatment did not affect BP (127±3 vs. 128±3mmHg), aortic nitrite (861±16 vs. 1004±174 μM/g) content and the development of atherosclerosis in L-NAME treated apoE-KO mice.

In conclusion, our findings indicate that Ang-(1-7) improves atherosclerosis via Mas receptor activation. Moreover, these effects seems to mediated through a NO-dependent mechanism as Ang-(1-7) failed to affect atherosclerosis in L-NAME treated mice.
Abstract P160: Role of Uncoupling Protein 2 in Stroke Susceptibility of Stroke-prone Spontaneously Hypertensive Rat
Authors: Rubattu Speranza , Cotugno Maria , Bianchi Franca , Di Castro Sara , Stanzione Rosita , Busceti Carla , Marchitti Simona , Nicoletti Ferdinando , Volpe Massimo
Published: July 2021

Mitochondrial dysfunction causes severe cellular derangements potentially underlying tissue injury and consequent diseases. Evidence of a direct involvement of mitochondrial dysfunction in hypertensive target organ damage is still poor.

The gene encoding Uncoupling Protein 2 (UCP2), a inner mitochondrial membrane protein, maps inside stroke QTL/STR1 in stroke prone spontaneously hypertensive rat (SHRSP). We explored the role of UCP2 in stroke pathogenesis of SHRSP. Male SHRSP, stroke resistant SHR (SHRSR) and reciprocal STR1/congenic rats were fed with stroke permissive Japanese style diet (JD). A group of SHRSP received JD plus fenofibrate (150 mg/kg/die). Rats were sacrificed at stroke occurrence. Additional SHRSR and SHRSP rats were sacrificed at 1, 3, 6, 12 months of age upon regular diet. SBP, BW, proteinuria, stroke signs were monitored. Brains were used for molecular analysis (UCP2 gene and protein expression, Nf-kB protein expression, oxidative stress quantification) and for histological analyses.

As a result, brain UCP2 expression was reduced to 20% by JD only in SHRSP (showing 100% stroke occurrence by 7 weeks of JD). Fenofibrate protected SHRSP from stroke and upregulated brain UCP2 (+ 100%). Congenic rats carrying STR1/QTL showed increased (+100%) brain UCP2 expression, as compared to SHRSP, when resistant to stroke, and, viceversa, decreased (-50%) brain UCP2 levels, as compared to SHRSR, when susceptible to stroke. Brain UCP2 expression progressively decreased with aging only in SHRSP, down to 15% level at one year of age (when SHRSP showed spontaneous stroke). Both brain Nf-kB expression and oxidative stress levels increased when UCP2 expression was downregulated, and viceversa. Histological analysis showed both ischemic and haemorrhagic lesions at stroke occurrence.

Our results highlight a role of UCP2 in stroke predisposition associated to hypertension in an animal model of complex human disease.
Abstract P168: ChIP-seq Analysis of Genomic Binding Sites for the Transcription Factor Fosl2 in Kidney Collecting Duct Cells
Authors: Jung Hyun Jun , Raghuram Viswanathan , Chou Chung-Lin , Knepper Mark
Published: July 2021

Kidney collecting duct principal cells play a key role in blood pressure regulation and water balance in part through the action of vasopressin. We integrated data from prior proteomics and transcriptomics studies of cultured mouse mpkCCD cells using Bayes’ Rule to rank transcription factors (TFs) likely to be involved in vasopressin-mediated transcriptional regulation. The top ranked TF was the AP-1 component Fos-like 2 (Fosl2) and the third ranked TF was Sox4. We carried out ChIP-seq analysis (HiSeq 2000 sequencer) to identify genomic sites of Fosl2 binding using an antibody successfully employed for ChIP-seq in the Mouse ENCODE Project. mpkCCD cells treated with the vasopressin analog dDAVP or vehicle were separately analyzed. The success of the immunoprecipitation of Fosl2 was confirmed by western blotting and LC-MS/MS of the immuno-precipitated material (ChIP-MS). ChIP-MS identified two different Fosl2 peptides, but there were no peptides corresponding to other AP-1 proteins. ChIP-MS also identified Bub1b, a nuclear protein kinase known to phosphorylate histones, as a Fosl2-interacting protein. Fosl2-binding sites successfully mapped to genes by ChIP-seq included a Fosl2 binding site, centered at 87 bp upstream from the transcription start site [TSS] of Sox4, that was strongly downregulated in response to dDAVP, n=2; confirmed by ChIP-PCR. TF binding site prediction (Genomatix) identifies putative Sox4 binding sites in vasopressin-regulated genes beta-ENaC (-822 bp from TSS) and aquaporin-2 (-515 bp from TSS). These data provide an initial step in identification of the vasopressin-regulated transcriptional network in renal collecting duct cells.
Abstract P194: A Novel Signalosome in the Alpha1a-Adrenergic Receptor and its Genetic Variant Signaling Pathway
Authors: Babaeva Ekaterina , Gradinaru Irina , Schwinn Debra A , Oganesian Anush
Published: July 2021

Objectives
: Human α
1
adrenergic receptors (AR), members of G protein-coupled receptor superfamily (GPCR) regulate blood pressure via smooth muscle cell (SMC) proliferation and vasoconstriction. We showed that α
1a
AR-G247R (247R) genetic variant, identified in a hypertensive patient, constitutively couples to β-arrestin1/MMP/EGFR transactivation pathway leading to hyperproliferation in fibroblasts, cardiomyoblasts and SMCs. A scaffolding protein spinophilin (SPL) serves as a docking site for many regulatory proteins including RGS2 (negative
R
egulators of
G
-protein
S
ignaling). SPL also binds 3
rd
intracellular loop of some GPCRs.

Hypothesis:
differential interaction of 247R or α
1a
AR-WT (WT) with SPL/RGS2/β-arrestin signalosome mediates unique signaling of 247R and hyperproliferation in cardiovascular cells.

Methods:
Receptor-protein interactions were determined by co-immunoprecipitation from HEK293 cells expressing HA-α
1
ARs and full length Myc-SPL, its fragments or Flag-RGS2. Protein levels were analyzed by Western. SPL knockdown or RGS2 overexpression was performed using SPL-specific or scrambled siRNA or Flag-RGS2.

Results:
Our results reveal that in SMCs or cardiomyoblasts 247R but not WT upregulates endogenous SPL. SPL exhibits stronger (~2-fold) interaction with WT compared to 247R or α
1b
, recruits RGS2 and inhibits receptor signaling. In contrast, weak SPL-247R interaction diminishes RGS2 inhibitory effect permitting hyperproliferation of 247R cells. SPL knockdown inhibits 247R-induced proliferation by allowing RGS2 directly bind to 247R as observed with RGS2 overexpression. Overexpression of SPL with RGS2 restores hyperproliferation suggesting that in 247R cells SPL binds RGS2 preventing RGS2-247R interaction.

Conclusions:
We present SPL/RGS2/β-arrestin as a novel signalosome responsible for α
1a
AR-247R genetic variant triggered hyperproliferation in different cardiovascular cells. We reveal that SPL regulates α
1a
AR signaling by differentially binding WT or 247R receptors and recruiting RGS2 protein to the receptor. These novel findings unravel critical roles of SPL and RGS2 in α
1
AR signaling, as well as identify SPL as a potential novel target for treatment of α
1
AR-mediated cardiovascular disorders.
Abstract P200: Endothelial Estrogen Receptor Alpha Does Not Protect Female Mice Against Western Diet Induced Vascular Stiffness
Authors: Manrique Camila , Lastra Guido , Haertling Dominic , DeMarco Vincent , Aroor Annayya , Jia Guanghong , Ramirez-Perez Francisco I , Martinez-Lemus Luis , Karas Richard , Chambon Pierre , Sowers James
Published: July 2021

Background:
Women with obesity, insulin resistance and type 2 diabetes mellitus (T2D) lose the cardiovascular disease protection normally afforded by female sex hormones, but the underlying mechanism(s) remain unknown. Increases in vascular stiffness occur with aging, but conditions of insulin resistance such as obesity and T2D are characterized by accelerated development of this phenomenon. Under physiological conditions, vascular estrogen signaling via estrogen receptor alpha (ERα) increases endothelial bioavailable nitric oxide which decreases stiffness. Nevertheless, in conditions of insulin resistance, the effects of ERα signaling may be deleterious.

Methods:
We used a novel rodent model lacking ERα in the endothelial cells (ECERαKO). The genomic region encompassing exon 3 of the ERα gene was flanked by loxP sites. ECERαKO mice were generated by crossing ERα doubled floxed mice with Cad-Cre+ mice (VE-Cadherin promoter driving expression of Cre-recombinase). Female ECERαKO mice and littermates were fed a high fructose/high sucrose (Western diet - WD) for 8 weeks. The WD diet consisted of 60% fat and 20% sucrose. At the end of the intervention period, mice underwent in vivo and ex vivo assessment of vascular stiffness.

Results:
The absence of EC ERα did not impact whole body insulin sensitivity (examined by HOMA-IR). Females lacking the endothelial specific ERα had less vascular stiffness when assessed in vivo via aortic pulse wave velocity than the littermates fed with a WD (3.43 ± 0.184 m/s vs. 4.080 ± 0.172 m/s, p<0.05). Similarly, ex vivo evaluation of aortic endothelial cell stiffness using atomic force microscopy (AFM) revealed increased stiffness in the females with intact EC ERα when compared with ECERαKO females (1.91 ± 0.60 kPa vs. 13.09 ± 2.61 kPa ) (p<0.05). Resistant vessel (femoral artery) also revealed less stiffness (decreased modulus of elasticity) in ECERαKO mice fed a WD.

Conclusion:
Endothelial ERα does not protect females from vascular stiffness induced by a WD. Indeed, the present data suggest a predisposition toward protection of rodent lacking ERα in conditions of insulin resistance.
Abstract P211: Normo-aldosteronemic Aldosterone-producing Adenoma: Immunochemical Characterization and Diagnostic Implications
Authors: Rossi Gian Paolo , Gioco Francesca , Fassina Ambrogio , Gomez-Sanchez Celso E
Published: July 2021

Background:
Primary Aldosteronism (PA), the most common form of endocrine hypertension, is usually identified by a high aldosterone-renin ratio (ARR) alongside elevated plasma aldosterone concentration (PAC) values. Normo-aldosteronemic PA is also being diagnosed when only the high ARR was driven by low renin because PAC was normal. However, whether this entity truly exists remains contentious since most such cases did not undergo surgical confirmation and even when adrenalectomy was performed no demonstration of an aldosterone-producing adenoma (APA) could be obtained at immunohistochemistry.

Case Description:
In 2003 a young lady presented with severe hypertension, low plasma renin activity (PRA), but consistently normal PAC values. She undertook a magnetic resonance that revealed a small adenoma on the right side, but adrenal vein sampling (AVS) showed left lateralization of aldosterone secretion. Therefore, she underwent left laparoscopic adrenal surgery, which determined complete normalization of blood pressure and PRA long term follow up (12 years). In 2014 the development of novel monoclonal antibodies for the human aldosterone synthase CYP11B2 and 11β-hydroxylase CYP11B1, allowed us to immunochemically characterize the resected adrenal gland. Double immunostaining for CYP11B2 and CYP11B1 showed a small CYP11B2-positive adenoma, thus unequivocally proving the presence of an APA.

Conclusions:
This case provides compelling evidence for the existence of normo-aldosteronemic APA and suggests that many cases that we dismiss as “low renin-essential hypertension” might instead have an undetected surgically curable APA.
Abstract P213: Western Diet May Modulate Kidney Injury and Albuminuria Differentially in Female Mice Deficient in Mr in the Endothelium versus the Smooth Muscle
Authors: Nistala Ravi , Simpson Ruth , Habibi Javad , Aroor Annayya , Garro Mona , DeMarco Vince , Hayden Melvin , Sowers James , Whaley-Connell Adam
Published: July 2021

Activation of the mineralocorticoid receptor (MR) has been implicated in kidney injury and precipitation of proteinuria. In this regard, diet induced obesity (DIO), a condition of MR activation is characterized by increase in kidney injury and proteinuria. DIO and other conditions of MR activation also manifest vascular dysfunction that may play a role in kidney injury and proteinuria. Vascular dysfunction may be endothelial or smooth muscle mediated. Moreover, MR signaling in the endothelium versus smooth muscle may be important in vascular function. Data from the Jaffe lab and our preliminary data show that deficiency of smooth muscle and endothelial MR plays a protective role from vascular dysfunction such as increased pulse wave velocity and stiffness. However, the role of endothelial specific versus smooth muscle specific MR in kidney injury and proteinuria is not known. Hence, we hypothesized that deficiency of endothelial and smooth muscle specific MRs (ECMRKO and SMMRKO) will protect the mice from Western diet-fed (high fat/high sucrose, WD) kidney injury and proteinuria. We fed female ECMRKO/SMMRKO and their littermate controls WD for 16wks and collected urine and performed imaging, molecular and morphological analyses. We observed significantly less proteinuria in the ECMRKO mice fed WD when compared to their littermates (2.4mg/mg vs. 3.5mg/mg creatinine) (p<0.05), however there was no change in the SMMRKO mice fed a WD when compared to their littermates. Furthermore, we observed significantly less impairment in aortic/renal pulse wave velocity and stiffness in both the ECMRKO/SMMRKO models. Western blots showed that there was a tendency to suppression of MR protein in the ECMRKO on WD. This suppression of MR expression was contemporaneously observed with decreased phosphorylation of ribosomal protein S6 along with reduction in membrane localization suggesting endothelial MR may regulate S6 activation. In summary, our study suggests endothelial specific MR may mediate kidney injury in conditions of MR activation and a lesser role for smooth muscle specific MR.
Abstract P225: Time of Food Intake is an Important Determinant of Blood Pressure Circadian Rhythm
Authors: Su Wen , Lutshumba Jenny , Guo Zhenheng , Gong Ming C
Published: July 2021

Blood pressure (BP) exhibits a 24-hour rhythm. Loss of BP oscillation is associated with significantly higher risk of target organ injuries. However, the mechanisms underlying the BP circadian rhythm remain incompletely understood. While light is a well-established prominent external cue that entrains intrinsic clock and circadian rhythm, recent studies indicate food intake is also an important external cue that can potently entrain intrinsic clocks especially those in peripheral tissues. However, whether BP circadian rhythm is affected by the time of food intake is unknown. If yes, via what mechanisms: are peripheral clocks and vascular functions involved? To address these specific questions, we used 12-14 weeks old male Per2::LUCIFERASE knock-in mice and investigated the effects of a two-week long, 10 hours light phase time restricted feeding (TRF, food only available from ZT2 to ZT 12) on BP circadian rhythm, clock gene oscillations and vascular contractile function. In the TRF group, food intake was initially decreased but gradually recovered to the ad libitum level by day 5. TRF did not alter body weight but significantly decreased non-fasting blood glucose. The BP was monitored continuously using radiotelemetry from 2 days prior to until 14 days after the TRF. Interestingly, the normal 24 hour BP oscillating rhythm transformed to a 12 hour oscillating rhythm by day 3 after TRF. Using LumiCycle, we investigated the clock gene Per2 expression in various isolated tissues. We found that the phase of Per2 luciferase oscillations was significantly shifted in liver and suprachiasmatic nucleus (SCN) containing brain slices, but was not significantly changed in aorta or mesenteric arteries. The oscillation amplitude of Per1 at mRNA level was suppressed in mesenteric arteries. Interestingly, the isometric contractile responses to high potassium depolarization, alpha1 agonist phenylephrine, and 5-HT were significantly suppressed in the abdominal aorta isolated from TRF group compared to those from ad libitum feed group. In summary, our results demonstrate that the time of food intake is an important determinant of blood pressure circadian rhythm. Moreover, time of food intake affects vascular clock gene oscillations and function.
Abstract P237: Dysregulation of Hypothalamic mTORC1, Sympathetic Nerve Activity and Vascular Reactivity in the Hypertensive Obese Prone Rats
Authors: Oltman Christine L , Muta Kenjiro , Dake Brian L , Morgan Donald A , Rahmouni Kamal
Published: July 2021

Neurogenic mechanisms play a major role in obesity-induced increase in sympathetic nerve activity (SNA) and arterial pressure, but the molecular pathways involved remain ill defined. Mechanistic target of rapamycin complex 1 (mTORC1) signaling in the hypothalamus has emerged as a critical molecular regulator of SNA, vascular function and arterial pressure. To analyze the status of hypothalamic mTORC1 signaling in obesity we compared the phosphorylated levels of ribosomal protein S6, a downstream effector of mTORC1, in the brain between obesity prone (OP) and obesity resistant (OR) rats. Body weight was elevated (P<0.05) in OP rats (763±22 g) relative to OR rats (575±19 g). OP rats also had higher fat mass. Interestingly, OP rats exhibited increased phospho-S6 in the mediobasal hypothalamus including the arcuate nucleus, but not in other nuclei such as the paraventricular nucleus, the supraoptic nucleus or the subfornical organ. Next, we assessed the hemodynamic and sympathetic parameters in OP and OR rats. Radiotelemetry systolic arterial pressure was greater in OP rats (133±1 mmHg) compared to OR rats (119±2 mmHg) at 8 weeks of age and remained elevated at 42 weeks of age. Ganglionic blockade with hexamethonium produced a dose-dependent decrease in arterial pressure in both the OP and OR rats, but the response was more pronounced (P<0.05) in OP rats. Direct SNA recording revealed elevated (P<0.05) renal and splanchnic SNA in OP rats (86±3 and 55±6 spikes/sec, respectively) compared to OR rats (48±2 and 22±4 spikes/sec). Using ultrasound Doppler, we found that OP rats have altered regional blood flows. Sodium nitroprusside-induced dilation was attenuated and phenylephrine-evoked constriction was potentiated in the hindquarters vasculature of OP rats relative to OR rats. However, there were no differences in the renal, mesenteric or abdominal aorta vascular beds. Acetylcholine (ACh)-mediated relaxation was impaired in isolated coronary arteries from OP rats (relaxation to 10 μM ACh: 41±8% in OP rats vs 67±10% in OR rats, P<0.05). These studies raise the possibility that overactivation of hypothalamic mTORC1 signaling contributes to the altered hemodynamic, sympathetic and vascular functions associated with the obesity prone phenotype.
Abstract P242: Role of Human Renal Proximal Tubule Sodium Bicarbonate Cotransporter NBCe2 (SLC4A5) in Salt Sensitivity of Blood Pressure
Authors: Gildea John J , Carlson Julia M , Hanh Tran T , Bigler Wang Dora , Xu Peng , Kemp Branden A , Langouet-Astrie Christophe J , McGrath Helen E , Carey Robert M , Jose Pedro A , Felder Robin A
Published: July 2021

The sodium bicarbonate cotransporter NBCe2 (encoded by SLC4A5) partially regulates renal tubular sodium bicarbonate transport. Hypothesis: since SLC4A5 single nucleotide polymorphisms (SNPs, rs10177833 and rs7571842) are associated with salt sensitivity of blood pressure, the gene product, NBCe2, would be involved with the etiology of human salt sensitivity. NBCe2 was localized in freshly fixed renal tissue and in primary and immortalized RPT cell (RPTC) cultures from tissue or isolated from urine. Basal expression of NBCe2 mRNA and protein was not different between RPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, total transcellular sodium transport, NHE3 protein expression, and Cl-/HCO3- exchanger activity were higher in SLC4A5 HV than WT RPTCs (WT: 8.6±1.2 mM NaCl n=6, 5207.1±386.4 RFU n=36, 0.265±0.006 pH unit/min, n=33 respectively; VS HV: 14.75±0.7 mM NaCl n=4, 6946.2±500.4 RFU n=48, 0.314±0.018 pH unit/min n=35 respectively, p<0.01). Aberrant sodium transport was even more evident after increasing intracellular sodium, which resulted in increased NBCe2 mRNA, NBCe2 protein and bicarbonate transport in HV RPTCs compared to WT (WT 146% ± 24% , 109% ± 4.7%, 89% ± 4.5%, respectively, VS HV 214% ± 23%, 128% ± 5.7%, 141% ± 4.8%, respectively N=8-12, p<0.05). RPTCs carrying HV variants showed increased binding of HNF4A to SLC4A5 DNA, which was blocked by two HNF4A antagonists. Assays in RPTCs isolated from urine showed increased bicarbonate-dependent pH recovery in RPTCs from salt-sensitive subjects who are HV for SLC4A5. NBCe2 under high sodium is hyper-responsive in RPTCs carrying SLC4A5 HV through an aberrant HNF4A-mediated mechanism.
Abstract P247: Tumor Necrosis Factor-alpha Receptors (Type 1 & Type 2) are Differentially Expressed in Renal Tissues During Chronic Dietary Intake of High Salt and Angiotensin II Treatment
Authors: Majid Dewan S , Prieto Minolfa C , Castillo Alexander
Published: July 2021

Tumor necrosis factor-alpha (TNF-α) exerts natriuresis that is mediated by its’ receptor type 1 (TNFR1) while its’ receptor type 2 (TNFR2) is involved in mediating inflammatory renal injury. To determine the differential roles of these receptors in angiotensin II (AngII) induced salt-sensitive hypertension and associated renal injury, protein expressions of TNFR1 and TNFR2 were examined in mice (n=6-7 in each group) chronically treated with or without AngII (25 ng/min; implanted minipump) for 4 weeks which were fed either normal (NS; 0.4% NaCl) or high salt (HS; 4% NaCl) diets. Systemic blood pressure (SBP) in these mice was measured by tail-cuff plethysmography and 24 hour urine collections were made using metabolic cages at the start and at the end of treatment period when the kidneys were harvested after sacrificing the mice with euthanasia. Immuno-histochemical analysis of TNFR1 and TNFR2 proteins in renal slices was performed by measuring the staining area as well as the intensity of receptors’ immunoreactivities using NIS Elements Software (Nikon), which allowed the semi-quantitation of positive staining and the intensity of these proteins. The results were expressed in percent area of positive staining and the relative intensity. HS intake alone did not alter mean SBP (HS; 77±1 vs NS; 76±3 vs mmHg) but it caused an exaggeration of AngII induced increases in mean SBP (AngII+HS; 104±2 vs AngII+NS; 95±2 mmHg). The area of TNFR1 staining was higher in HS (6.0± 0.9 vs 3.2 ±0.7%; P<0.05) than NS group but it was not significantly different between AngII+HS (5.0± 0.7%) and AngII+NS groups (6.3±0.7%). Similar qualitative differences were also observed in relative intensity in protein expressions. TNFR2 immunoreactivity was minimal in NS and HS groups but it was high in AngII+NS group and even greater in AngII + HS group. These data suggest that the increases in TNFR1 activity due to HS alone facilitate salt excretion that results no change in SBP in response to HS intake. However, such HS induced increases in TNFR1 activity was compromised in elevated AngII condition causing more salt retention and thus, exaggerated hypertensive response. On the other hand, HS induced increases in TNFR2 activity in elevated AngII condition facilitates enhanced renal injury response.
Abstract P257: High Fat Diet Induces Metabolic Syndrome-Like Phenotype Associated with Adverse Micro- and Macro-Vascular Remodeling
Authors: Trask Aaron J
Published: July 2021

Metabolic diseases such as type 2 diabetes (T2DM), hypertension, and metabolic syndrome (MetS) have been associated with vascular disease, and we have previously demonstrated vascular-bed-specific remodeling in both mouse and porcine models of T2DM and MetS. The aim of this study was to determine whether high-fat diet would induce MetS-associated adverse micro- and macro-vascular remodeling and mechanics in mice. Three week old male C57BL/6J mouse siblings were randomized to receive either a normal low-fat (LFD: 10% fat) or high-fat (HFD: 60% fat) diet for 20 weeks (n=7-10 per group). HFD induced a MetS-like phenotype characterized by increased body weight (LFD: 28.6±0.7 vs. HFD: 43.4±0.8 g,
p
<0.0001), increased mean arterial pressure (LFD: 65±3 vs. HFD: 91±2 mmHg,
p
<0.0001), increased plasma insulin (LFD: 106±39 vs. HFD: 368±54 pg/mL,
p
<0.001), and transient increases in fasting blood glucose. Passive pressure myography of septal coronary resistance microvessels (CRMs) revealed reduced internal (LFD: 151±11 vs. HFD: 113±7 μm at 125 mmHg,
p
<0.05) and external diameters, increased wall/lumen ratio (LFD: 5.5±0.6 vs. HFD: 7.8±0.5 at 125 mmHg,
p
<0.01) and reduced incremental modulus of elasticity (LFD: 7.9x10
6
±1.7x10
6
vs. HFD: 4.5x10
6
±0.6x10
6
dynes/cm
2
at 125 mmHg,
p
<0.01) in mice fed HFD. Adverse CRM remodeling was associated with reduced coronary flow at baseline and under hyperemic conditions, which reduced coronary flow reserve (LFD: 7.3±0.5 vs. HFD: 5.5±0.5,
p
<0.05). Aortic pulse wave velocity was increased (LFD: 0.31±0.02 vs. HFD: 0.36±0.01 cm/ms,
p
<0.05) and significantly correlated with the increased blood pressure (r=0.67, p<0.01). These data demonstrate that 20 weeks of a high-fat diet induces an early MetS-like pathophysiological state that is associated with vascular-bed-specific remodeling and alterations in vascular biomechanics. Furthermore, the presence of adverse vascular remodeling in the presence of an early MetS-like phenotype, but not overt MetS (i.e. in the presence of sustained elevation in fasting blood glucose), may suggest the presence of underlying sub-clinical disease during the early progression of metabolic syndrome.
Abstract P258: Essential Hypertension Induces Early Functional and Structural Vascular Aging in Small Resistance Arteries
Authors: Virdis Agostino , Bruno Rosa Maria , Duranti Emiliano , Taddei Stefano
Published: July 2021

We evaluated cross-sectionally whether vascular remodeling is physiologically present in normal aging, and whether hypertension causes an acceleration of the aging process for vascular function and structure.

40 essential hypertensive patients (EH, age 44.9±13.2 years; BP, 157±8/99±3 mmHg) and 36 normotensive control individuals (Ctrl, age 44.7±12.7 years; BP: 128±7/80±4 mmHg) underwent laparoscopic surgery with subcutaneous adipose tissue biopsy. Small resistance arteries were studied by pressure micromiography. Endothelium-dependent and -independent vasodilation were evaluated by dose-response curve to Acetylcholine (ACh) and sodium nitroprusside (SNP). Maximum %inhibition by L-NAME on response to Ach was calculated. Structural alterations were assessed by media-lumen ratio (M/L).

EH showed a reduced vasodilation to Ach (P<0.001), but not to SNP, compared to Ctrl. In both groups, %inhibition by L-NAME on response to ACh was inversely related to age (EH, r:-0.75; P<0.0001; Ctrl, r:-0.49; P<0.0001). NO availability was significantly reduced in EH as compared to Ctrl for each age group (<30 years: 22±6% vs 30±9%, P<0.05; 31-45 years: 17±3% vs 30±3%, P<0.0001; 46-60 years: 9±4% vs 21±6%, P60 years: 4±3% vs 13±3%, P<0.05). Age-hypertension interaction (Repeated measures ANOVA) was not significant (p= 0.25). EH showed an increased M/L (P<0.001) compared to Ctrl. In both groups, M/L was positively related to age. (EH, r:0.82; P<0.0001; Ctrl, r:0.50; P<0.0001). M/L was similar in EH and Ctrl for individuals <30 years, but greater in EH than Ctrl for the other age groups (31-45 years: 6.5±0.4% vs 5.6±0.4%, P<0.0001; 46-60 years: 7.4±0.5% vs 5.8±0.2%, P60 years: 7.9±0.3% vs 6.3±0.5%, P<0.0001). There was a significant age-hypertension interaction (Repeated measures ANOVA p= 0.0009).

In small resistance arteries, aging is characterized by progressive reduction in NO availability and increased M/L. In hypertensive patients, NO availability is early reduced in comparison to Ctrl, but the progression rate with age appears to be similar. Conversely, structural alterations are influenced by hypertension only after 30 years of age, but the progression rate with age is steeper in the presence of hypertension.
Abstract P617: Angiotensin II-induced Hypertension And Vascular Injury Is Mediated By Gamma/delta T Cells
Authors: Caillon Antoine , Oneeb Rehman Mian Muhammad , Barhoumi Tlili , Paradis Pierre , Schiffrin Ernesto L.
Published: July 2021

Objective:
Both innate antigen presenting cells and the adaptive immune system have been shown to play a role in the development of hypertension. Nevertheless, the T cell subset involved in the pathophysiology of hypertension remains unclear. There is a small subset of “innate-like” T cells expressing gamma/delta T cell receptor (TCR) rather than the alpha/beta TCR that could play a role in bridging between the innate and adaptive immune systems. However, it is unknown whether gamma/delta T cells contribute to development of hypertension.

Method/Results:
Thirteen to 15 week-old male C57BL/6 wild-type and Tcrd-/- mice, which are devoid of gamma/delta T cells, were infused or not with angiotensin (Ang) II (490 ng/kg/min, SC) for 7 or 14 days (n=4-9). Telemetric blood pressure, mesenteric artery endothelial function and vascular remodeling by pressurized myography and spleen T cell profile by flow cytometry were evaluated. Fourteen days of Ang II increased systolic blood pressure (167±4 vs 125±2 mmHg, P≤0.01) in wild-type compared to control mice. The frequency of gamma/delta T cells (6±1% vs 3±1%, P≤0.05) and activated (CD69+) gamma/delta T cells (11±1% vs 7±1%) was increased after 7 days of Ang II, and 7 days later were respectively unchanged or further increased (24±2% vs 10±1%) in wild-type compared to control mice. Ang II decreased mesenteric artery relaxation responses to acetylcholine (51±5% vs 88±3%, P≤0.01) and increased media/lumen (5±1 vs 3±0%, P≤0.01) in wild-type mice compare to controls. No gamma/delta T cells were detected in Tcrd-/- treated or not with Ang II. All the above Ang II effects were abrogated in Tcrd-/- mice.

Conclusion:
These data suggest that gamma/delta T cells mediate Ang II-induced blood pressure rise and vascular injury. Gamma/delta T cells could be key immune cells bridging innate and adaptive immune responses during the development of hypertension.
Abstract P618: Foxp3+ Regulatory T cell Depletion Eliminates Ang II-Induced Hypertension Resistance in Female Mice
Authors: Pollow Dennis , Romero-Aleshire Melissa J , Uhrlaub Jennifer , Konhilas John P , Nikolich-Zugich Janko , Brooks Heddwen L
Published: July 2021

Compared to males, premenopausal females are resistant to the development of Ang II hypertension. In males, Ang II induces hypertension, in part, through mechanisms requiring T effector lymphocytes. Recently, our lab has demonstrated that females can prevent the T lymphocyte-dependent increase in blood pressure (SBP and MAP) and expression of pro-inflammatory cytokines in the kidney in response to Ang II infusion. Because Foxp3
+
T regulatory cells suppress the pro-inflammatory and hypertensive actions of T effector cells, we sought to determine whether Foxp3
+
T regulatory cells contribute to this resistance in females. Premenopausal (8 week old) 129SVE female mice were infused with Ang II (800ng/kg/min, 14d) and received 4 doses of the anti-CD25 antibody PC-61 to transiently deplete Foxp3
+
T regulatory cells (every 84 hours beginning 12 hours prior to Ang II infusion, 250μg/dose, i.p., vehicle control). Blood pressure was measured before and after Ang II infusion via non-invasive tail cuff. Ang II induced a significant increase in systolic blood pressure in Foxp3
+
-depleted mice, while resistance was retained in vehicle-treated mice (Con Δ5 ± 5mmHg, Ang II Δ10 ± 7mmHg, PC-61 Δ28 ± 9
*
mmHg,
*
p<0.05 vs Con). Flow cytometric analysis demonstrated that PC-61-treatment significantly reduced the number of Foxp3
+
splenic T cells compared to control (Con 1.7x10
6
cells, Ang II 2.3x10
6
cells, PC-61 8.3x10
5*
cells,
*
P<0.05 vs Con) without changing CD3
+
and CD4
+
T cell counts. The number of Foxp3
+
T cells residing in the kidney was also significantly reduced by PC-61 (Con 1,152 ± 368 cells, Ang II 686 ± 389 cells, PC-61 210 ± 35
*
cells,
*
P<0.05 vs Con). Quantitative real-time PCR demonstrated that whole kidney expression of MCP-1 and ENaC alpha were significantly increased in Foxp3
+
-depleted mice (MCP-1- Con 1.0 ± 0.1, Ang II 1.6 ± 0.4, PC-61 1.8 ± 0.2
*
; ENaC-α- Con 1.0 ± 0.1, Ang II 1.6 ± 0.2, PC-61 2.1 ± 0.1
*
,
*
P<0.05 vs Con). These data suggest that the anti-inflammatory Foxp3
+
T regulatory cells play a significant role in mediating the resistance to Ang II hypertension in premenopausal female mice, and may influence renal inflammation and sodium retention during chronic Ang II infusion.
Abstract P628: Angiotensin 1-7 Mas Receptor and Dopamine D1 receptor Interaction as a Novel Natriuretic Mechanism in Rat Kidneys
Authors: Diaz Diaz Andrea C , Lokhandwala Mustafa F , Jafri Faraz , Banday Anees A
Published: July 2021

Evidence to date suggests that a positive interaction between natriuretic factors promotes sodium excretion to maintain sodium homeostasis and blood pressure. Although the involvement of renal dopamine D1 receptor (D1R) in promoting sodium excretion is well established; the role of Angiotensin (Ang) 1-7 Mas receptor (MasR) is not clear. Here we provide evidence for a functional interaction between these two renal G protein-coupled receptors which suggests that natriuretic response to Ang 1-7 via MasR is dependent on D1R activation. Male Sprague Dawley rats of comparable weight and age were infused with Ang 1-7, MasR antagonist DPro, D1R agonist SKF38393 and D1R antagonist SCH23390. Blood pressure was monitored throughout the experimental procedure and none of the infused drugs affected the pressure. Animals infused with saline alone served as controls. Infusion of Ang1-7 caused significant natriuresis and robust diuresis compared to saline. SKF38393 infusion also induced significant natriuresis and diuresis when compared to saline infusion. Both natriuretic and diuretic response to Ang 1-7 was blocked by Dpro and SCH23390. However, Dpro failed to block SKF38393 response. Concomitant infusion of SKF38393 and Ang 1-7 did not show a cumulative natriuretic or diuretic effect when compared to SKF38393 or Ang 1-7 infusion alone. FENa (%), control (saline): 0.30 ± 0.09; Ang 1-7: 1.03 ± 0.21; Ang 1-7 plus Dpro: 0.49 ± 0.11; Ang 1-7 plus SCH23390: 0.36 ± 0.10; SKF38393: 0.83 ± 0.16; SKF38393 plus SCH23390: 0.41 ± 0.09; SKF38393 plus Dpro: 0.82 ± 0.17; Ang 1-7 plus SKF38393: 1.06 ± 0.21. These data suggest that Ang 1-7 via MasR causes natriuresis which is dependent on D1R activation. On the other hand, renal D1R-mediated sodium excretion is independent of MasR. This study is a paradigm shift as these data identify a novel functional unidirectional interaction between renal MasR and D1R which deviates from commonly known receptor-receptor interaction.
Abstract P639: Attenuation of Renal Inner Medullary Circadian Clock Gene Expression in Response to High Salt Intake is Dependent on the Endothelin B Receptor
Authors: Speed Joshua S , Hyndman Kelly A , Kasztan Malgorzata , Johnston Jermaine G , Young Martin E , Pollock Jennifer S , Pollock David M
Published: July 2021

Our lab has recently shown that ETB deficient (ETB def) rats have a time of day dependent impairment in their ability to excrete a Na+ load. These observations suggest an interaction between renal ETB receptors and circadian mechanisms that regulate renal tubular Na+ transport and excretion. Given that knockout of the circadian clock gene Bmal1 reduces blood pressure in mice, we hypothesized that a high salt intake impairs the clock mechanism in the renal inner medulla in an ETB dependent manner. Transgenic control (Tg con) or ETB def rats were fed normal (NS, 0.8% NaCl) or high (HS, 4% NaCl) salt for two weeks. In one group, rats were euthanized every 4 hours beginning at zeitgeber time 0 (lights on) for tissue collection (and subsequent assessment of circadian clock genes), while in a second group of rats urine was collected in 12-hour intervals (active vs. inactive). Consistent with our hypothesis, we observed that HS abolished the normal oscillation in Bmal1 expression in the renal inner medulla of Tg con rats, and effect not observed in ETB def rats. Interestingly, renal production of ET-1, was significantly higher during the active period vs. inactive period in both NS (3.6±1.1 vs. 0.8±0.2 pg/12hr respectively) and HS (9.2±4.1 vs. 1.6±0.3 pg/12hr respectively) fed Tg con rats. There was no time-of-day-dependent difference in ET-1 excretion in ETB def rats on NS (6.6±2.2 vs. 4.6±1.7 pg/12hr respectively), although this pattern was restored in ETB def rats fed HS (2.2±1.0 vs. 9.2±2.5 pg/12hr inactive vs. active). Taken together, these data indicate that an increase in renal ET-1/ETB activation in response to HS modulates inner medullary clock gene expression to promote renal Na+ excretion.