bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 04: Growth Arrest Specific 6 And Axl Signaling Coordinate Endothelial Cell And Immune Cell Activation To Promote Inflammation And Hypertension.
Authors: Van Beusecum Justin P , Barbaro Natalia , Smart Charles D , Ao Mingfang , Patrick David M , De La Visitacion Nestor , LOPERENA Roxana , Xiao Liang , Shibao Cyndya A , Harrison David G
Published: August 2021

Endothelial cells (ECs) activated by hypertensive (10%) cyclical stretch releases factors including IL-6 and hydrogen peroxide that stimulate the conversion of human monocytes to an intermediate inflammatory phenotype. A novel subset of DCs in humans has been identified that express Axl and Sigelc-6
+
(AS DCs) which drive T cells proliferation and produce inflammatory cytokines. The interplay between ECs and AS DCs in hypertension is unkown. We assessed AS DCs by flow cytometry in normotensive (n=23) and hypertensive (n=11) subjects and found a significant increase in AS DCs in hypertensive compared to normotensive subjects (297 ± 73 vs. 108 ± 26/ml;
p
=0.0304). When moncoytes were exposed to human aortic endothelial cells (HAECs) undergoing 10% stretch, the formation of AS DCs was markedly enhanced compared to 5%. The ligand for Axl is growth arrest specific 6 (GAS6), and we found that 10% HAEC stretch caused a 50% increase in the release of GAS6 by ECs comapred to 5%. We knocked down either EC GAS6 or Axl using siRNA and either of these abrogated the ability of ECs to promote AS DC formation. Using flow cytometry to analyze venous ECs that had been harvested from 23 volunteers to quantify EC activation and GAS6 secretion in vivo, we found a positive association between GAS6 and ICAM-1 (R
2
=0.39,
p
=0.0012). We found a positive association between pulse pressure and plasma GAS6 (R
2
=0.25,
p
=0.0079) ands systolic blood pressure and GAS6 (R
2
=0.19, p=0.0025) in volunteers. We found that plasma GAS6 is increased in Ang II hypertension and that either genetic deletion or pharmacological inhibition of Axl lowered blood pressure in reposne to Ang II and reduced renal inflammation. To investigate the role of immunological vs. stromal Axl in vivo, we perfomed bone marrow transplant studies and found that both Axl
WT/WT
->Axl
-/-
and Axl
-/-
->Axl
W/WT
had a significant reduction in blood pressure by 20 mmHg compared to the Axl
WT/WT
-> Axl
WT/WT
control. These data show that both immunological and stromal Axl contribute to hypertension and inflammation and GAS6/Axl signlaing may be a novel therapeutic target in this disease.
Abstract 11: Single Cell Eccite-seq Analysis Reveal An Important Role Of Monocyte-specific Nlrp3 Inflammasome For Salt-sensitivity Of Hypertension In Humans
Authors: Pitzer Ashley L , Sahinoz Melis , Raddatz Michael , Wanjalla Celestine , Pakala Suman , Elijovich Fernando , Laffer Cheryl L , Ikizler Alp , Kirabo Annet
Published: August 2021

Salt sensitivity of blood pressure is an independent predictor of death due to cardiovascular events. Diagnosis of salt-sensitivity is not feasible in the clinic, making it difficult to investigate therapeutic strategies. We hypothesized that NLRP3-inflammasome and IL-1β production in monocytes plays a role in salt-sensitive hypertension. We phenotyped salt-sensitivity of blood pressure using an acute inpatient Weinberger protocol of an isocaloric high salt diet and rapid intravenous salt-loading, followed by low salt diet and furosemide-induced salt-depletion. Ambulatory blood pressure was continuously monitored and averaged for the days of salt-loading and salt-depletion. Blood samples were obtained at baseline, salt-loading, and after salt-depletion. Median age was 54 years (44-55), 3 of the 5 subjects were female, screening systolic blood pressure was 140 mmHg (134-148), diastolic blood pressure was 88 mmHg (84-99), and BMI was 35 kg/m
2
(30-39). Using cell hashing and ECCITE-seq analysis, we profiled transcriptomes in multiple immune cell types using antibody-derived tags (ADTs). UMAP clustering of different cell types were identified by ADTs including monocyte markers CD14 and CD16. Interestingly, UMAP visualization of CD14+ and CD16+ clusters indicated a greater decrease in CD14+ clusters after salt-depletion in the salt-resistant subjects than the salt-sensitive; however the salt-sensitive subjects had a greater decrease in CD16+ clusters than the salt-resistant group after both salt-loading and salt-depletion. These data were confirmed using flow cytometry. Unlike in salt-resistant participants, we found that within monocyte clusters, salt-sensitivity was associated with down regulation of the inflammasome components NLRP3 (0.386 ± 1.18 vs. 0.197 ± 0.778) and IL-1β (0.858 ± 2.32 vs. 0.159 ± 0.925) following salt-depletion. Using flow cytometry, we found
Δ%
isoLG+ CD14+/CD16+ monocytes correlated with salt-sensitivity of blood pressure (r=0.88, 95% CI, p=0.05). These results suggest that the inflammasome and monocyte activation are dynamically regulated by dietary salt in vivo and can serve as a potential diagnostic biomarker for salt-sensitivity of blood pressure.
Abstract 12: The Role Of Memory Gamma Delta T Cells In Hypertension And Vascular Damage
Authors: Comeau Kevin D , Paradis Pierre , Schiffrin Ernesto L
Published: August 2021

Background:
We recently demonstrated that γδ T cells participate in the pathogenesis of hypertension. Evidence also suggests that memory T cells may develop during an initial hypertensive episode, sensitizing mice to develop hypertension to further mild hypertensive challenges. However, whether memory γδ T cells develop and play a role in hypertension remains unknown. Our objective is to determine if memory γδ T cells sensitize mice to develop hypertension in response to a mild hypertensive challenge.

Methods:
Ten-12-week-old C57BL/6J mice were exposed or not to a hypertensive challenge (490 ng/kg/min angiotensin II (Ang II), SC) for two weeks, followed by a two-week washout period, and then infused with a subpressor dose of Ang II (140 ng/kg/min Ang II, SC) for two weeks. Blood pressure was measured via telemetry and central, effector, and resident memory γδ T cells were profiled by flow cytometry.

Results:
Mice exposed to the first hypertensive challenge had a higher systolic blood pressure than the sham group at the end of the subpressor hypertensive challenge (149±6 vs. 122±3 mmHg,
P
<0.001). After 14-days of Ang II infusion, effector memory γδ T cells increased 5.2-fold in the mesenteric artery perivascular adipose tissue (PVAT, 1.25±0.37% vs. 0.24±0.12%,
P
<0.05), and 1.8-fold in the mesenteric lymph nodes (mLN, 1.49±0.03% vs. 0.82±0.15%,
P
<0.05) compared to sham treated mice. After repeated Ang II infusion, central memory γδ T cells decreased by 57% in the aortic PVAT (6.79±1.46% vs. 15.69±2.87%,
P
<0.05), and by 22% in the mLN (0.18±0.01% vs. 0.23±0.01%,
P
<0.05) compared to control mice.

Conclusion:
An initial exposure to a hypertensive stimulus sensitizes mice to develop hypertension to a subsequent subpressor hypertensive challenge and results in the development of memory γδ T cells.
Abstract 26: IL-17 Causes Hypertension, Reduced Fetal Weight And Natural Killer Cell Activation In The Absence Of T Cells
Authors: Fitzgerald Sarah J , Hogg James , Deer Evangeline , Campbell Nathan , Herrock Owen , Lemon James , Amaral Lorena , Cornelius Denise C , Cockrell Kathy , Ibrahim Tarek , Turner Ty , Lamarca Babbette A
Published: August 2021

Preeclampsia (PE) is characterized by new onset hypertension (HTN), intrauterine growth restriction (IUGR), multi-organ dysfunction, and is associated with increased inflammatory cytokines, such as interleukin 17 (IL-17). More recent studies demonstrate a role for mitochondrial (mt) dysfunction/mtROS in the pathogenesis of PE. Although we have shown T helper cells from a rat model of PE cause HTN and mt dysfunction the causative factors for mt dysfunction are still being identified. In addition, we have shown that IL-17 cause HTN, IUGR and activate natural killer (NK) cells, and cause mt dysfunction in pregnant Sprague Dawley rats. However, in our previous studies we couldn’t differentiate the effect of activated TH cells versus IL-17 to cause these characteristics of PE. The athymic nude rat model lacks mature T cells but does have other components of the immune system, and will thus allow us to examine the role of IL-17 in the absence of TH cells in the pathophysiology of PE. We hypothesize that in the absence of T cells IL-17 induces HTN, NK cell activation and IUGR which is associated with renal and placental mt dysfunction during pregnancy. To test our hypothesis, IL-17 (150 pg/day) was infused via osmotic minipumps inserted on gestation day (GD) 14. Blood pressure (MAP) and mt function were measured on GD19 and were compared to untreated pregnant (NP) athymic nude rats. In response to IL-17; MAP increased from 95±4mmHg in NP(n=6) to 115±2 in NP+IL-17(n=6) (p<0.001); pup weight decreased from 1.46±0.2 g in NP (n=6) to 0.98±0.07g in NP+IL-17 (n=6) (p<0.05); NK cell activation increased from 0±0 %lymphocytes in NP (n=3) to 0.4±0.1% lymphocytes in NP+IL-17 rats (n=6). Interestingly, placental mtROS reduced 54% fold compared to NP and renal mtROS reduced 51.2% compared to NP. ATP production increased from 15.53±1.6 pmol of O2/sec/mg in NP (n=3) to 105.5±91 pmol of O2/sec/mg in NP+IL-17 (n=3) in the placenta, and from 1196±460 pmol of O2/sec/mg in NP (n=4) to 2016±951 pmol of O2/sec/mg NP+IL17 (n=4) in the kidney. These results show that although IL-17 induces HTN, IUGR, and NK cell activation independent of T cells, T cells are necessary for reduced mitochondrial function observed in PE and in rat models of placental ischemia.
Abstract 37: Loss Of BK
Ca
- IP3R Functional Coupling Underlies Vascular Hypercontractility In Spontaneously Hypertensive Rats
Authors: Niloy Sayeman Islam , Jarajapu Yagna , O'Rourke Stephen , Sun Chengwen
Published: August 2021

The cellular mechanisms underlying enhanced vascular contractility in hypertension are still not fully clear. Recent studies have demonstrated that functional coupling between the large-conductance Ca
2+
-activated K
+
(BK
Ca
) channel in the cell membrane and inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum is involved in the regulation of intracellular Ca
2+
in vascular smooth muscle cells (VSMC) and vascular contractility. Here, we examined the role of BK
Ca
-IP3R coupling in vascular hypercontractility in Spontaneously Hypertensive Rats (SHR), as compared with Sprague Dawley (SD) rats. In pressurized mesenteric arteries, paxilline (1μM), a selective BK
Ca
channel blocker, significantly increased norepinephrine (1μM)-induced vasoconstriction in both SHR (% initial diameter: 71.6±6.8% [95.9±13 of 132.8±6.2 μm] and 65.0±4.8% [80.6±7.7 of 123.6±3.9 μm] in arteries treated with vehicle and paxilline respectively, n=4, P<0.05) and in SD rats (% initial diameter: 84.2±7.9% [108.4±12.3 of 128.5±2.5 μm] and 60.5±0.2% [77.4±0.7 of 127.9±0.6 μm] in arteries treated with vehicle and paxilline respectively, n=3, P<0.05). Paxilline-induced increases in vasoconstriction were significantly greater in arteries of SD as compared with SHR rats, suggesting that BK
Ca
channels are involved in hypercontractility in SHR. In inside-out patch-clamp, BK
Ca
channels in VSMCs isolated from mesenteric arteries of SHR and SD rats displayed identical calcium and voltage sensitivity. We next examined the effect of the selective IP3R-agonist, Adenophostin A, on BK
Ca
current density in VSMCs, using patch-clamp whole-cell mode. The results demonstrated that Adenophostin A (5μM) significantly increased BK
Ca
current density in VSMCs from both SHR (from 41.9± 5.7 to 61.5±9.1 pA/pF; n=6 cells, p<0.05) and SD rats (from 29.9 ± 8.0 to 92.6 ± 9.1 pA/pF, n=5 cells, P<0.01). There was no significant difference in basal BKca current density between SHR and SD rats; however, the Adenophostin-induced elevation in BK
Ca
current density was significantly diminished in VSMCs of SHR as compared with SD rats. In conclusion, the results indicate that loss of functional coupling between BKca and IP3R is involved in vascular hypercontractility in SHR arteries.
Abstract 46: Vascular Endothelium-derived Endothelin 1 Promotes Salt-induced Kidney Inflammation In Male But Not Female Mice
Authors: Molina Patrick , Biswal Sara , Pollock Jennifer S , De Miguel Carmen , Edell Claudia
Published: August 2021

Endothelin 1 (ET-1), potent vasoactive and pro-inflammatory peptide, plays a criticalrole in hypertension and end-organ damage. The inflammatory response to high saltintake displays sex differences; however, if a sex difference exists in vascular ET-1inducing salt-induced kidney inflammation is unknown. We aimed to assess the role ofendothelium-derived ET-1 in promoting kidney inflammation in male and female micefed high salt. Aged-matched male and female vascular endothelial cell ET-1 knockout(VEET KO) and control floxed ET-1 (VEET
fl/fl
) mice were fed 4.0% NaCl chow (HSD).After 3 weeks of HSD, kidney myeloid and lymphoid cell populations and their functionalstatus were examined by flow cytometry. In response to HSD, male VEET
fl/fl
mice hadsignificantly higher kidney TH17 cell numbers than female VEET
fl/fl
mice (IL-17A
+
CD4
+
cell absolute count, male vs. female: 229.6±27.7 vs. 84.9±17.2, p=0.0003, n=6-10/group), indicating a substantial sex-dependent difference in kidney TH17 cellabundance. Lack of endothelium-derived ET-1 resulted in 72% reduction in renal TH17cell number in males (VEET
fl/fl
vs. VEET KO: 229.6±27.7 vs 64.0±64, p=0.0002), but notin females (p>0.05). Frequency of kidney IL-17A
+
CD4
+
cells was not different betweengenotypes in females or between male and female VEET
fl/fl
mice. However, male VEETKO mice displayed 57% less renal IL-17A
+
CD4
+
cells than male VEET
fl/fl
(respectively,0.6±0.071 vs. 1.4±0.2, p=0.0055), and 59% reduction compared to female VEET KOmice (1.4±0.2, p=0.0052). Assessment of the effects of vascular ET-1 on the innateimmune response showed that activated kidney resident macrophages(F4/80
hi
CD11b
+
CD64
+
) were increased in male vs. female VEET
fl/fl
(MFI, respectively,2080±105 vs. 1672±25, p=0.0002). Lack of endothelium-derived ET-1 significantlyreduced CD64 abundance in males (p=0.012), but not females (p>0.05), whencompared to VEET
fl/fl
. In conclusion, endothelium-derived ET-1 is critical in promotingrenal innate and adaptive inflammatory responses in males in response to high salt, andimportant male-female differences exist in the manner by which vascular ET-1 regulateskidney inflammation in this setting. Funded by NIH F31 HL151264-0 to PAM,P01HL136267 to JSP, and K01HL145324 to CDM.
Abstract 49: Supine Versus Seated Positions On The Detection Of Orthostatic Hypotension And Its Association With Fall Risk And Orthostatic Symptoms
Authors: Juraschek Stephen P , Appel Lawrence J , Miller Edgar R
Published: August 2021

Background:
Hypertension trials that monitor orthostatic hypotension (OH) compare standing to seated blood pressure (BP) rather than supine BP. We determined the impact of a supine vs seated position on OH prevalence and its relationship with fall risk and orthostatic symptoms.

Methods:
The Study to Understand Fall Reduction and Vitamin D in You (STURDY) was a randomized trial testing the effects of vitamin D3 dose on falls in adults age ≥70 years at higher risk of falls. OH was determined at baseline, 3, 12, and 24 months with each of 2 protocols: (1) seated to standing and (2) supine to standing. OH was defined as a drop in systolic or diastolic BP of at least 20 or 10 mm Hg. Participants were asked about orthostatic symptoms in the past month. Falls were ascertained via daily fall calendar, ad hoc reporting, and scheduled interviews.

Results:
Among 522 participants with 953 OH assessments (mean age 76 ± 5 years, 42% women, 18% Black), mean baseline BP was 129 ± 18/68 ± 11 mm Hg. Mean BP increased 3.4/2.6 mm Hg after sitting, but decreased -3.7/-0.7 mm Hg after being supine. OH was detected in 2.2% of seated vs 14.8% of supine assessments. Supine OH better predicted falls (HR 1.60; 95% CI: 0.98, 2.61;
P
=0.06) than seated OH (HR 0.70; 95% CI: 0.30, 1.60;
P
=0.39), although both were non-significant (
Figure
). While seated OH was not associated with orthostatic symptoms, supine OH was associated with a greater risk of fainting, blacking out, seeing spots, room spinning, and headache in the prior month (
P
-values of 0.048 to 0.002).

Conclusions:
Supine OH was more prevalent and appeared to better predict falls and orthostatic symptoms than seated OH. These findings support a supine protocol for OH in clinical practice.
Abstract 55: Hiv Viral Proteins Elevate Blood Pressure And Impairs Endothelial Function In Resistance Arteries Via Sex Specific, Immune And Nox1-dependent Mechanisms In Mice.
Authors: Kress Taylor
Published: August 2021

Thanks to the onset of combination antiretroviral therapy (cART), patients living with HIV live longer but experience accelerated rates of hypertension. However, the etiology of HIV-associated hypertension remains ill defined, specifically the respective contributions of repressed viral infection and cART treatment. Herein, we took advantage of a transgenic model (Tg26) of repressed viral infection to investigate the contribution of HIV viral proteins to hypertension. Quantification of inflammatory cytokines revealed elevated circulating TNFα levels but also high aorta and intraperitoneal immune cells TNFα in male Tg26 mice leading to the hypothesis that HIV-associated hypertension involves immune-derived TNFα secretion and endothelial dysfunction in males and females Tg26 mice. Blood pressure (BP) was measured via radio telemetry and vascular reactivity studies performed via wire myography. BP analysis revealed increased mean arterial pressure (MAP: male: WT=112.3±1.3 vs Tg26=121.9±4.0 mmHg/ female: WT=110.6±3.01/ Tg26=120.3±6.9 mmHg) and heart rate in both sexes (P<0.05). However, blockade of TNFα action with etanercept restored BP and aortic endothelial function in male Tg26 mice only. Along with an increase in TNFα, male Tg26 aortas showed infiltration of Tcells and proinflammatory cytokines IL-18, and IL-6, which were not seen in females. However, scavenging of reactive oxygen species with the selective NOX1 inhibitor GKT771 restored aorta endothelial function in both sexes. A contributor to hypertension is impaired endothelial relaxation in resistance arteries. Mesenteric arteries showed impaired endothelial relaxation to acetylcholine in both male and female Tg26 mice (P<0.05) with no impairment in smooth muscle cell relaxation. This phenotype was reproduced in WT mice transplanted with Tg26 bone marrow (BM) and remarkably reversed in Tg26 mice receiving WT BM supporting a clear role for immune cells in endothelial dysfunction. Collectively, these data indicate that HIV-related hypertension involves immune and NOX1-dependent endothelial dysfunction in both sexes but TNFα-dependent mechanisms in males only providing evidence of sex specific mechanisms.
Abstract 60: Perirenal Adipose Hypertrophy In A Congenic LH Rat: A Role For
C17h6orf52
Authors: Clark Karen C , Holl Katie L , Reho John J , Grobe Justin L , Kwitek Anne E
Published: August 2021

Central obesity, high blood pressure, dyslipidemia, and insulin resistance are a collection of cardiovascular and metabolic risk factors that form the basis of the Metabolic Syndrome (MetS) and represent a major public health burden worldwide. The phenotypes that define MetS are all highly heritable, but their genetic complexity necessitates the use of animal models to tease apart novel pathways. The Lyon Hypertensive (LH) rat is a well-characterized model of MetS, exhibiting profound differences in features of MetS compared to its metabolically healthy control, the Lyon Normotensive (LN) rat. To understand the genomic causes of MetS, we developed a congenic rat model, where a portion of LN chromosome 17 is introgressed on the LH genomic background. Male and female LH congenic (CON) rats and LH controls were phenotyped for a variety of MetS characteristics, including body growth and composition by nuclear magnetic resonance (NMR), metabolic rate (Promethion system), and adipose tissue collection and histological examination. There were significant decreases in body weight in the CON rats of both sexes compared to LH. We also found significant female-specific increases in body fat and decreases in metabolic rate. Tissue collection revealed the source of the increased adiposity in the female CON rats was specific to perirenal white adipose tissue (PWAT) and was further explained by significant hypertrophy in those adipocytes. Genome resequencing of the parental strains identified a gene,
C17h6orf52
, as a strong contender underlying the phenotype differences in the congenics, with predicted amino acid changes and the loss of Nr2f2 transcription factor binding sites.
Abstract MP11: Decreased Expression Of Ace2 Is Associated Urine Derived Human Renal Proximal Tubule Cells From Inverse Salt Sensitivity Participants Cultured In Low Salt Conditions
Authors: Gildea John J , Xu Peng , Schiermeyer Katie , Yue Wei , Felder Robin A
Published: August 2021

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. Inverse salt sensitivity (ISS) is the paradoxical increase in blood pressure of individuals to a low sodium diet. Our group previously reported decreased expression of dopamine type 2 receptor (D
2
R), increased expression Aminopeptidase N, and increased Ang II dependent sodium transport in human urine derived renal proximal tubule cells isolated from ISS participants. In an attempt to understand the increased Ang II sensitivity demonstrated in ISS cells, we examined angiotensin converting enzyme 2 (ACE2), a membrane associated enzyme involved in the metabolism of Ang II. Urine derived renal proximal tubule cells grown and immortalized from ISS participants were compared to cells from salt resistant (SR) participants cultured in iso-osmotic media with low salt (LS, 90 mM NaCl) normal salt (NS, 140 mM NaCl) and high salt (HS, 190 mM NaCl). Cells were incubated in LS, NS, and HS media with and without losartan (LOS,1 μM) overnight (18 hours) and ACE2 expression levels determined by in-cell western blot. A monoclonal antibody specific to an extracellular epitope of ACE2 was used as the primary antibody and an Alexa-647 anti-mouse secondary antibody. ACE2 expression was only reduced in ISS cells in LS condition (28.7±2.1 % reduction, ISS LS vs SR LS, N=4 per group, p<0.05). Addition of losartan completely blocked the decrease in ACE2 expression in low salt conditions in ISS in urine derived human renal proximal tubule cells. No other changes in ACE2 expression were found between ISS and SR in either NS or HS conditions and with or without losartan. In conclusion, a decreased expression of ACE2 in ISS urine proximal tubule cells could explain the previously reported increased sensitivity of ISS cells to Ang II by increasing the half-life of Ang II under low salt conditions.
Abstract MP12: The
Mef2c
Transcription Factor Regulates The Renin Cell Identity
Authors: Guessoum Omar E , Kupkova Kristyna , Sheffield Nathan , Sequeira Lopez Maria Luisa , Gomez Roberto A
Published: August 2021

Introduction:
The Renin-Angiotensin-System is essential to maintain blood pressure and fluid electrolyte homeostasis. Because precise regulation of expression and release of
renin
is critical for survival, understanding the molecular regulation of the renin cell identity is a vital area of study. Advances in epigenetics have enabled finer dissection of chromatin factors which maintain the identity of the renin cell. By studying genes with heightened accessibility profiles that are unique to the JG cell, we now have the capacity to unravel the determinants of the renin cell identity.

Hypothesis:
That transcription factors central to the governance of renin cell identity can be identified through the Assay for Transposase Accessible Chromatin (ATAC-seq) differential accessibility analysis.

Methods:
Native renin cell ATAC-seq was compared to existing ENCODE ATAC-seq datasets from 40 other cell types to define regions/peaks which characterize the JG program. Peaks with high intensity and ≥2-fold increase in signal were selected for Motif analysis to search for transcription factors (TFs) whose consensus sequence is enriched in those regions. Identified TFs were then selected for validation by
in-situ
hybridization and conditional deletion in renin cells.

Results:
1) The
Mef2c
transcription factor was identified as having a consensus sequence in regulatory regions unique to the JG cell. It has clear expression in RNA-seq of renin cells (65 transcripts per million, n=3) and a predicted binding site in the
renin
gene. These results were validated by
in-situ
hybridization where signal localized at the JG area was detected in concordance with our
in-silico
results. 2) We generated
Mef2c
conditional knockout animals using our
Ren1d-Cre
mouse to study the effect in renin expression and identity. These mice displayed reduced renin immunostaining at the JG area and a 40% reduction in renin mRNA expression by qPCR from kidney cortices relative to wild-type (n=2, preliminary data).

Conclusions:
Our studies identified
Mef2c
as a TF target which likely has an essential role in maintaining and preserving renin cell identity. Experiments involving transcriptomics and epigenomics are ongoing to understand the changes wrought by
Mef2c
deletion in renin cells.
Abstract MP16: Perivascular Adipose Tissue Inflammation Impairs Microvascular Endothelial Function In People Living With HIV
Authors: Wang Dan , Wang Cheng , Kassaye Seble , Kurmar Princy , Verbesey Jennifer , Wilcox Christopher S
Published: August 2021

Introduction and hypothesis:
We had reported that people living with HIV (PLWH) have microvascular endothelial dysfunction and increased ROS. We now tested the hypothesis that perivascular adipose tissue (PVAT) could enhance oxidative stress and inflammation and impair the function of subcutaneous microarterioles (SMAs) in PLWH.

Methods:
SMAs were obtained from young, virally-suppressed HIV-infected subjects (n=11) or matched controls (n=11). Subjects were without associated CVD risk factors. Microvascular reactivity and PVAT function were accessed by myograph from isolated subcutaneous vessels with or without PVAT of skin biopsy.

Results:
The HIV-infected group had significantly (P<0.05) increased adipose MDA, TNFα, IL-1α, leptin and reduced adiponectin. The PVAT-denuded SMAs from the HIV group had significantly (P<0.05) impaired acetylcholine-induced endothelium-dependent relaxation factor (EDRF, 26±4 vs 38±3%) and NO activity (0.35±0.03 vs 0.58± 0.07 Δfluoresence unit) and significantly (P<0.05) increased contraction to U-46,619 (200±8 vs 141±7%) and endothelin 1 (ET1, 167±12 vs 118±17%) and ROS generation (0.32 ± 0.06 vs 0.1 ± 0.03 (E/DHE fluoresce unit). PVAT enhanced EDRF (50±4 vs 38±3 %) and NO (0.84 ±0.1 vs 0.58±0.07 Δfluoresence unit ) only in controls (P<0.05). The reduction of U46,619 anti-contractivity by PVAT is decreased in HIV (48±7 vs 85±9%, P<0.05).

Conclusion:
HIV-infected individuals have intrinsic vascular defects from ROS augmented by extrinsic vascular defects from adipose inflammation that impairs the beneficial microvascular PVAT signaling. Therefore, targets for potential prevention of cardiovascular morbidity in PLWH should include the elimination of ROS and inflammation in both microvessels themselves and the surrounding extravascular PVAT.
Abstract MP28: UBR1 And BRCC36 Regulate ACE2 Ubiquitination And Deubiquitination In Ang-II Induced Hypertension.
Authors: Lakkappa Navya , Berdasco Clara , Filipeanu Catalin , Lazartigues Eric D
Published: August 2021

UBR1 and BRCC36 regulate ACE2 Ubiquitination and Deubiquitination in Ang-II induced Hypertension.
Navya Lakkappa
1
, Clara Berdasco
1
, Catalin Filipeanu
2
, Eric Lazartigues
1
1. Pharmacology, LSUHSC, New Orleans, LA; 2. Pharmacology, Howard University, Washington, DC. ACE2 is undergoing Ang-II-mediated internalization, ubiquitination and degradation, contributing to hypertension. Using proteomics and bioinformatic analysis, we identified UBR1 and BRCC36 and evaluated their role in ACE2 ubiquitination in hypertension. Following gonadectomy, blood pressure (BP, telemetry) was recorded in C57BL6/J mice (3-month-old, n=10/group) infused with Ang-II (490 ng/Kg/min/4 weeks sc). UBR1, BRCC36 and ACE2 expression was assessed in heart and kidneys (capillary western). Ang-II infusion induced a significant mean BP increase in all groups (sham males: 133 ±0.6; castrated: 141 ±0.6; sham females: 134 ±0.7; ovariectomized: 130 ±0.3 mmHg; n=6, one-way ANOVA, P<0.001). UBR1 levels were similar between sexes while cardiac BRCC36 was 2-fold higher in females (P<0.001). Ang-II-mediated hypertension led to a 4-fold increase in UBR1 in males heart and kidneys (P<0.001) and a 2-fold increase in females (P<0.01) while gonadectomy blunted this effect by 50%. Castration did not affect BRCC36 levels while ovariectomy reduced them by 5-fold. Males infused with Ang-II showed no significant change in BRCC36 expression while hypertensive females had a dramatic reduction (5-fold, P<0.001) in the heart. Overall, UBR1 was associated with a reduction and BRCC36 was associated with an increase of ACE2 levels. Endothelial cells (HAEC) exposed to Ang-II (100 nM for 4h) had a 2-fold increase in UBR1 and a 10-fold reduction in ACE2 (n=3: one-way ANOVA, P< 0.01). Complete knockdown of UBR1 resulted in a 10-fold increase in ACE2 levels (n=3: one-way ANOVA, P<0.05). In HEK293T cells transfection with BRCC36 reversed the Ang-II effects (72533.5 ±4432.2
vs.
42204.2 ±4338.7; FU/min/μg protein, n=3, one-way ANOVA, P<0.05). Together, we show that ACE2 expression in hypertension is differentially regulated by URB1 and BRCC36 and depends on sex hormones. Inhibition of UBR1 and upregulation of BRCC36 could be a new therapeutic strategy to prevent ACE2 ubiquitination in hypertension.
Abstract MP29: A Cloned Anti-aminopeptidase N Scfv Antibody Blocks Enzymatic Activity And Rescues A Low Sodium Phenotype In Proximal Tubule Cells Isolated From Inverse Salt Sensitive Participants
Authors: Gildea John J , Xu Peng , Yue Wei , Felder Robin A
Published: August 2021

Inverse Salt Sensitivity (ISS), defined as the paradoxical increase in blood pressure of individuals on a low sodium diet compared to a high sodium diet, may be associated with the increase in mortality and morbidity found in individuals on a low sodium diet. Our group has previously found that urine derived human renal proximal tubule cells (RPTC) isolated from ISS participants express higher Aminopeptidase N (APN) protein than cells isolated from salt resistant (SR) participants. An anti-Aminopeptidase N (APN) hybridoma was used construct a single chain variable fragment (scFv) into a bacterial expression system. The purified anti-APN scFv bound to live human renal proximal tubule cells, detected using an Alexa594 directly labelled anti-myc monoclonal antibody by immunofluorescent confocal microscopy. The anti-APN scFv was then tested to determine if it blocks APN enzymatic activity. We have previously shown that under low sodium conditions the amount of reduced-glutathione as determined by live cell fluorescence staining with the redox sensitive dye, monochlorobimane (mBCl), is decreased only in ISS cells under low sodium conditions at the two-hour time point. We hypothesized that the decreased mBCl signal may be due to the increased APN expression and activity altering the Ang II/Ang III ratio and reducing the Ang III activation of the AT
2
R. Low sodium reduced the mBCl signal in ISS (-29.2±4.3%, ISS vs SR, N=3 per group, p<0.05) and the addition of the anti-APN scFv at 1 ug/ml completely blocked the mBCl signal back to levels found in SR in normal salt conditions. In order to verify that the full rescue of this ISS specific response is due to enhanced Ang III – AT
2
R signaling, we next tried to block the effect of the anti-APN scFv by the addition of the AT
2
R antagonist, PD123319 (PD, 1 uM). Addition of PD alone to the ISS LS RPTCs did not significantly alter the mBCl signal, but when PD and anti-APN scFv are added to the LS treated ISS cells, there is complete reversal of the effect of anti-APN scFv alone (-38.1±3.9%, ISS anti-APN scFv + PD vs ISS anti-APN scFv, N=3 per group, p<0.05). The anti-APN scFv has potential therapeutic value by reducing APN enzymatic activity in ISS individuals and inducing the protective AT
2
R arm of the renin angiotensin system in low sodium conditions.
Abstract MP34: Collecting Duct Cells-derived Human Sprr Impairs The Antihypertensive Effects Of Losartan And Upregulates Erk1/2 And Aqp2 Expression
Authors: Arthur Gertrude , Poupeau Audrey , Nichols Kellea , Leachman Jacqueline , Loria Analia S , Osborn Jeffrey , Yiannikouris Frederique B
Published: August 2021

Recent studies showed that soluble prorenin receptor (sPRR) plays an important role in blood pressure regulation and in water balance. In rodent models, sPRR contributes to AngII production by increasing renin activity, systolic blood pressure (SBP) and aquaporin2 (AQP2)-dependent antidiuretic action. However, there is a gap of knowledge concerning the functional role of locally produced sPRR from the kidney. Therefore, we evaluated the kidney-derived human sPRR role in SBP control and fluid homeostasis. Human sPRR-Myc-tag transgenic mice were bred with mice expressing Hoxb7/Cre to selectively express human sPRR in the collecting duct (RHsPRR). RHsPRR and control (CTL) male mice were fed a standard diet for 10 months (n=8-11/group). Body weight and urine volume were examined and SBP measured by radiotelemetry. Western blot analysis depicted the presence of human sPRR-Myc-tag (28 KDa) in the cortex and medulla of RHsPRR male mice validating the humanized mouse model. Body weight did not change and 24hr-SBP was similar between CLT and RHsPRR mice (128±2 and 122±5 mmHg, respectively). However, the chronic response to losartan treatment was reduced in RHsPRR compared to CTL (ΔSBP: CTL: -9±3; RHsPRR: -5±1 mmHg, P<0.05). Kidney-derived human sPRR did not change GFR (838±75 vs 1088±163 μl/min/100g BW) and urinary vasopressin (0.62±0.21; 0.72±0.20 ng/mg creatinine), while modestly decreasing urine excretion rate by ~40% (CTL: 1.04±0.20; RHsPRR: 0.57±0.25 ml/day). Furthermore, RHsPRR mice had higher AQP2 protein expression in renal cortex (CTL: 0.24±0.07; RHsPRR: 4.11±0.70 AU, P<0.05) and medulla (CTL: 0.11±0.04; RHsPRR: 4.03±1.74 AU, P<0.05) than CTL mice. Kidney-derived human sPRR significantly increased phosphorylation of ERK 1/2 in the cortex compared to CTL (CTL: 5.4±1.0; RHsPRR: 9.2±1.4 AU, P<0.05), an MAPK involved in the regulation of water balance. In addition, RHsPRR mice showed increased plasma osmolality compared to CTL mice (CTL: 349±2; RHsPRR: 357±2 mOsm/kg, P<0.05). Overall, our data suggest that renal human sPRR could contribute to the increase in plasma tonicity by promoting the activation of ERK1/2-AQP2 pathway. Whether this signaling is associated with impaired antihypertensive effects of AT1R blockage remains under investigation.
Abstract MP36: Human Urinary Exosomes Contain Functional ACE2
Authors: Okechukwu Charles C , Pirro Nancy T , Chappell Mark C , Yamaleyeva Liliya M
Published: August 2021

The Ang II convertase and SARS-COV-2 co-receptor ACE2 is highly expressed on proximal tubules within the kidney. ACE2 is also present in urine and reportedly correlates with various renal pathologies that may reflect enhanced shedding of the peptidase through activation of ADAMs. Indeed, 95 kDa ACE2 is typically detected in urine consistent with a shorter, soluble form of the peptidase; however, the full-length, membrane-bound form of ACE2 (120 kDa) is also evident in urine which is difficult to reconcile with ACE2 shedding. To account for these isoforms, we evaluated ACE2 expression in exosomes isolated from human urine. Morning collections from males [50 to 64 years of age, non-smokers] were immediately processed for exosome isolation by cibacron blue binding of albumin followed by 0.2 μmicron filtration to remove microvesicles and apoptotic bodies, Amicon 100 kDa concentration, and ultracentrifugation (UC) to pellet exosomes. Analysis of the UC pellet fraction revealed the exosomal markers ALIX, CD63 and HSP70, as well as the proximal tubule peptidases neprilysin (NEP) and ACE2. Exosomal ACE2 content was 45 ± 11 ng/mL (mean ± SEM; N=5) by ELISA and exosomal activity hydrolyzed Ang II to Ang-(1-7) that was abolished by the ACE2 inhibitor MLN4760. Fluorescent nanotracking analysis (f-NTA) with Alexa Fluor antibodies and CellMask Deep Red membrane stain (CMDR) demonstrate a similar density of ACE2+ and NEP+ exosomes that were ~50% of total urinary exosomes (*P<0.05 vs. CD63+, N=3) while particle sizes were comparable and in the expected range of exosomes (100-150 nm). We conclude that human urinary exosomes express functional ACE2 which may originate from proximal tubule release.
Abstract MP37: Epigenetic Changes Following Maternal Gut Microbiota Improvement With Dietary Fibre Lead To Cardio-protection In The Offspring
Authors: Jama Hamdi , Dona Malathi I , Dinakis Evany , Nakai Michael E , Paterson Madeleine , Shihata Waled , Weeks Kate L , Farrugia Gabriella E , Salimova Ekaterina , Kaipananickal Harikrishnan , Okabe Jun , Mackay Charles , El-Osta Assam , Pinto Alex , Kaye David M , Marques Francine Z
Published: August 2021

Dietary fibre is fermented by the gut microbiota and protects against the development of cardiovascular disease (CVD) through the production of gut microbial metabolites. We hypothesised dietary fibre intake during pregnancy may prevent the development of CVD in the offspring via in utero epigenetic mechanisms. To investigate this, we fed C57BL/6J female mice diets high or low in resistant starches (‘high-fibre’ and ‘low-fibre’, respectively) during gestation. At 6-weeks of age, we performed single-cell RNA-sequencing in the offspring (n=8/group) or they were challenged with saline (sham) or angiotensin II (Ang II, 0.25mg/kg/day, n=18-23/group). Maternal diet resulted in a distinct gut microbial composition (
P
=0.001). This was still evident in the adult offspring, with high-fibre offspring having a different gut microbial colonisation (
P
=0.001), irrespective of sham/Ang II treatment. Maternal fibre intake significantly changed the cardiac cellular and molecular landscape and promoted differential gene signatures in the offspring. This included upregulation of genes associated with extracellular matrix production in the offspring from low-fibre mothers. When challenged with Ang II, low-fibre offspring developed increased cardiac hypertrophy (
P
=0.034) and fibrosis (
P
=0.01) compared to high-fibre offspring. This was accompanied with decreased ejection fraction (
P
=0.001) and increased left ventricular posterior wall thickness (
P=
0.017). These changes were independent of blood pressure. High-fibre offspring had decreased expression of natriuretic peptides (
Nppa
,
P
=0.03,
Nppb
,
P
=0.002) compared to low-fibre offspring. Chromatin-immunoprecipitation assay revealed decrease in H3-acetylation at the cis-regulatory region of
Nppa
gene in Ang II-treated high-fibre offspring (P=0.002), suggesting that maternal fibre intake influences the epigenetic changes of the
Nppa
gene in the offspring’s heart. Together, these data reveal maternal high-fibre intake leads to foetal epigenetic reprogramming, likely through maternal to foetal transfer of gut microbial-derived metabolites.
Abstract MP58: Tissue And Sex Specific Effects Of
Gstm1
Deletion In Mouse Models
Authors: Wang Yves , Nguyen Nhu , Nehrke Keith , Brookes Paul S , Le Thu H
Published: August 2021

The glutathione S-transferase (
Gst
) gene family encodes antioxidant enzymes. In humans, a common null allele deletion variant of
GST μ-1
(
GSTM1
) is highly prevalent across populations and is associated with increased risk and progression of various diseases. Using a
Gstm1
knockout (KO) mouse model, we previously showed that KO mice with angiotensin II-induced hypertension (HTN) have increased kidney injury compared to wild-type (WT) controls, mediated by elevated oxidative stress. In the same mouse model, we have recently reported that in a Langendorff-perfused cardiac ischemia-reperfusion injury (IRI) model, where damage is also mediated by oxidative stress, male KO hearts are protected while females are not. Here, we investigated the molecular mechanisms for this difference in male hearts. WT and KO mice of both sexes were studied at 12-20 weeks of age. Hearts were snap frozen at baseline and after 25 min of global ischemia, and kidneys were collected at baseline and 4 weeks following HTN induction. A panel of 18
Gst
genes were probed by qPCR from baseline hearts and kidneys of both sexes. Global metabolites were assayed using Metabolon, Inc. from hearts of both sexes and kidneys of males, at both baseline and diseased states. Analysis by qPCR (n = 3/group) showed that male, but not female, KO hearts had upregulation of other
Gst
s. In contrast, no significant differences between were found in male kidneys. Metabolomics (n = 6/group) detected 695 metabolites in hearts and 926 in kidneys. There were increases in several metabolites in KO vs. WT hearts including those with antioxidant properties. Notably, increases in carnosine and anserine were observed in KO male hearts but not in female hearts, while that of other antioxidant-related metabolites were observed in hearts of both sexes, but not in kidneys. HTN induced significant increases in metabolites in KO vs. WT kidneys in the pathways related to and linking methionine, cysteine, and glutathione, which were not observed in hearts. In this study, gene expression and metabolites suggest that the mechanisms compensating for the loss of GSTM1 are both tissue and sex specific. The resulting differences in antioxidant enzymes and metabolites may explain the unexpected protection for male
Gstm1
KO hearts in IRI.
Abstract MP59: Soluble Protein Oligomers Induce Endoplasmic Reticulum Stress In Mesenteric Resistance Arteries From Male And Female Mice
Authors: Waigi Emily W , Castaneda Thaddaeus R , Bearss Nicole R , Edwards Jonnelle M , Giovannucci David R , McCarthy Cameron G , Wenceslau Camilla F
Published: August 2021

Amyloid β proteins, including toxic soluble oligomers (SPOs) are not only found in the brain duringAlzheimer’s, but also in the peripheral vascular system. The precise mechanism linking increasedcirculating levels of SPOs and vascular dysfunction remains unknown. We hypothesized that SPOslead to endoplasmic reticulum (ER) stress, further release of SPOs and vascular injury. Mesentericresistance arteries (MRAs) from 14 weeks old, male and female C57BL/6 mice were used forvascular function. Agonists were acetylcholine and phenylephrine (1nM-10mM). In acuteconditions, SPOs (0.1μM) caused pathologically exacerbated endothelium-dependent vasodilationcompared to vehicle (F12 media) [Male: EC50: SPOs: -7.0 ± 0.1 (n=4), vs. Vehicle -6.6 ± 0.1 (n=7)p=0.03; Female: EC50: SPOs: -7.3 ± 0.06 (n=5) vs. Vehicle -6.7 ± 0.1 (n=6), p=0.001]. Thisphenotype was similar to the positive control tunicamycin (5mg/ml) [Male: EC50: Tunicamycin: -7.3(n=4), vs. Vehicle -6.6 (n=7) p=0.2; Female: EC50: Tunicamycin: -7.7 (n=4) vs. Vehicle -6.8 (n=5)p=0.04]. To determine whether SPO’s cause ER stress, arteries were treated with ER stressinhibitor 4-Phenylbutyric acid (2mM). The ER stress inhibitor prevented the exacerbatedvasodilation induced by SPOs showing SPOs trigger ER stress in acute conditions independent ofsex. To determine whether SPOs are a consequence of ER stress, arteries were incubated withtunicamycin in the presence of the SPO inhibitor K01-162 (10mM). Interestingly, K01-162 did notprevent the tunicamycin-induced exacerbated vasodilation in arteries from male mice. However,this response was decreased in arteries from female mice showing that inducing ER stress leadsto the release of SPOs, escalating a feed-forward mechanism of further SPO release. There wereno changes in vascular contraction with tunicamycin or SPOs irrespective of sex. ER stress wasconfirmed with anti-KDEL antibody staining, specific for ER resident chaperones Grp78/94 andvisualized with multiphoton fluorescent confocal microscopy. These results demonstrate that SPO’sexacerbate endothelium-dependent vasodilation acutely and may contribute to brain and peripheralvascular edema and loss of autoregulation observed during cardiovascular and Alzheimer’sdisease.
Abstract MP64: Estimated Impact Of Public Health System Reforms To Increase Hypertension Treatment Coverage In India
Authors: Marklund Matti , Cherukupalli Rajeev , Pathak Priya , Neupane Dinesh , Krishna Ashish , Wu Jason H , Matsushita Kunihiro , Appel Lawrence J
Published: August 2021

Background:
Approaches to scale up hypertension (HTN) treatment are needed in India, where only ~10% of individuals with HTN have controlled blood pressure.

Objective:
Estimate the current HTN treatment capacity of the public health system in India and model the effects of selected health system reform options.

Methods:
Using constrained optimization models, we estimated the HTN treatment capacity and salary costs of HTN-treating staff within the public health system; and simulated the potential effects of 1) increased workforce, 2) greater task sharing, and/or 3) reduced visit frequency (quarterly) vs the common practice of monthly visits for prescription refills.

Results:
An estimated 8% of all adults with HTN could be treated in the status quo (current number of health workers, no further task sharing, and monthly visits) (Figure). Treating 70% of adults with HTN with monthly visits without greater task sharing could require an additional 1.6 million staff, with ~200 billion ₹ (≈US$2.7 billion)) in additional annual salaries. Greater task sharing was estimated to allow the current workforce to treat ~25% of individuals with HTN with monthly visits. Quarterly visits (i.e., longer prescription periods) together with greater task sharing could allow the current workforce to treat ~70% of patients with HTN in India.

Conclusion:
Expanding HTN treatment coverage through workforce expansion alone will require substantial human and financial resources. The combination of greater task sharing and quarterly visits could increase the coverage of HTN treatment to ~70% of adults with HTN in India, without any expansion of the current workforce of the public health system.