bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 054: Prorenin Stimulates Firing Activity in Hypothalamic Neurons via Angiotensin II-Dependent and -Independent Mechanisms
Authors: Pitra Maria S , Feng Yumei , Stern Javier E
Published: July 2021

The brain RAS plays a central role in cardiovascular homeostasis by modulating sympathetic nerve activity and hormone secretion. Still, the relevance of intrinsic brain RAS function is questioned because renin levels are too low to have an impact on angiotensin II (AngII) formation. The discovery of the prorenin receptor (PRR) brought new support to the brain RAS involvement in neurohumoral regulation. However, the mechanisms underlying PRR-mediated actions remain unknown. We used whole cell patch-clamp electrophysiology in hypothalamic slices to examine the effects of prorenin (PR) on the excitability of magnocellular neurosecretory neurons (MCNs) of the supraoptic nucleus and parvocellular RVLM-projecting presympathetic neurons of the paraventricular nucleus (PVN-RVLM), which play integral roles in cardiovascular control, and have been implicated in the pathophysiology of neurogenic hypertension.

PR application (2.5nM) increased the firing activity of both MCNs (basal 1.3±0.4 Hz
vs
PR 3.3±0.7 Hz; p=0.0086) and parvocellular neurons (basal 0.5±0.1 Hz
vs
PR 2.1±0.4 Hz; p=0.0006), including a subset of PVN-RVLM neurons (basal 0.5±0.1 Hz
vs
PR 1.4±0.4 Hz; p=0.048). In MCNs, this effect was blocked by a PRR antagonist (
PRO20
; 250nM; basal 1.9±0.6 Hz
vs
PRO20 1.3±0.5 Hz; p=0.017), but persisted in the presence of the AT1 receptor blocker, losartan (50μM; basal 0.8±0.2 Hz
vs
losartan 2.3±0.5 Hz; p=0.0018). Conversely, PR effects on PVN neurons persisted with PRO20 (basal 0.9±0.2 Hz
vs
PRO20 1.6±0.3 Hz; p=0.013), but were largely (~75%) blunted with losartan (basal 0.2±0.04 Hz
vs
losartan 0.6±0.1 Hz; p=0.0193; Δ basal 1.6±0.4 Hz
vs
Δ losartan 0.4±0.1 Hz; p=0.0175). These results suggest that while PR actions are primarily PRR-mediated in MCNs, they are predominantly AngII-dependent in PVN cells. Lastly, PR effects were abolished in both cell types by dialyzing neurons with the Ca
2+
chelator BAPTA (10mM; MCNs: basal 0.6 ± 0.1 Hz
vs
BAPTA 0.3 ± 0.1 Hz; p=0.0277; PVN: basal 0.4±0.05 Hz
vs
BAPTA 0.5±0.2 Hz; p=0.6468).

Our results show for the first time that PR stimulates firing activity in both hypothalamic neurosecretory and presympathetic neurons, and support distinct AngII-independent and dependent signaling mechanisms in each neuronal population.
Abstract 064: 20-HETE Contributes to Vasoconstriction of Renal Microvessels and Sodium Retention in Cyp4a14-/- Mice, a Model of 20-HETE Dependent Hypertension
Authors: Gilani Ankit , Pandey Varunkumar , Zullo Joseph , Mishra Priyanka , Falck John R , Capdevila Jorge , Schwartzman Michal
Published: July 2021

20-HETE (20-Hydroxyeicosatetraenoic acid), is a cytochrome P450 (CYP) 4A-derived arachidonic acid metabolite. 20-HETE has been linked to both pro-hypertensive (via increased vasoconstriction, vascular remodeling and vascular injury of renal microvessels) and anti-hypertensive (inhibiting ion transport in the distal nephron) functions. In this study we examined the effect of 20-SOLA (2,5,8,11,14,17-hexaoxanonadecan-19-yl-20-hydroxyeicosa-6(Z),15(Z)-dienoate), a water soluble antagonist of the actions of 20-HETE on renal hemodynamics and sodium (Na) excretion in Cyp4a14 knockout (CYP4a14-/-) male mice. The CYP4a14-/- male mice display hypertension accompanied by increased vascular 20-HETE levels. Administration of 20-SOLA (10mg/kg/day in drinking water) normalized blood pressure (BP) in male Cyp4a14-/- mice at day 10 of treatment (124±1 vs. 153±2 mmHg in untreated male Cyp4a14-/- mice; p<0.05). The normalization of blood pressure was accompanied by transient increase in the urinary sodium excretion in the Cyp4a14-/- male mice (8.3±0.7 vs. 5.8±0.5 μmol/g body weight/day; p<0.05). Importantly, 20-SOLA increased glomerular filtration rate (GFR) of Cyp4a14-/- mice (2.38±0.05 vs. 1.88±0.18 μL/min/mg kidney weight, p<0.05) as opposed to no changes observed in the wild type (WT: (2.26±0.18 vs. 2.33±0.20μL/min/mg kidney weight). Evaluation of the renal blood flow (RBF) by laser Doppler flowmetry showed that treatment with 20-SOLA increased the RBF in Cyp4a14-/- mice by 12.3±4%, which remained unaltered in the WT. Additionally, the pressure-induced myogenic tone of isolated preglomerular microvessels was significantly elevated in Cyp4a14-/- mice; 20-SOLA treatment prevented the increase in myogenic responses. The natriuretic response to an isotonic saline loading challenge (10% of body weight, IP) was significantly attenuated in the Cyp4a14-/- mice as compared to the WT (35.5±2.8 vs. 57.4±8.3 percentage of Na load, p<0.05); this was corrected by 20-SOLA (61.7±5.7 percentage of Na load, p<0.05). These results confirm that 20-SOLA normalizes blood pressure of Cyp4a14-/- male mice and demonstrates that this is associated with increases in GFR, RBF and natriuresis.
Abstract 066: The Undescribed Protein Caskin2 Is a Novel Regulator of eNOS Phosphorylation and Systemic Blood Pressure
Authors: Mueller Sarah B , Gurley Susan B , Kontos Christopher D
Published: July 2021

Disruptions in the function of the quiescent endothelial cells (ECs) that line mature vessels can both result in and contribute to the progression of numerous cardiovascular diseases including hypertension, atherosclerosis, and disorders of vascular permeability. Despite recent attention, the signaling pathways that are active in quiescent ECs remain poorly characterized relative to those that regulate EC activation. In an effort to provide mechanistic insight into these pathways, we have characterized the previously undescribed protein Caskin2, which we hypothesize is a novel regulator of EC quiescence. Caskin2 is expressed in ECs throughout the vasculature, including the aorta, coronary arteries, and renal glomeruli. In vitro, Caskin2 promotes a quiescent EC phenotype characterized by decreased proliferation and increased resistance to apoptosis-inducing factors. Caskin2 knockout mice are viable and fertile. However, preliminary radiotelemetry measurements indicate that Caskin2 knockout (KO) mice have mildly elevated systemic blood pressure (BP). Compared to wild type (WT) littermates (n=8), Caskin2 KO mice (n=7) had increased mean arterial pressure (119+/-1 vs. 113+/-1, p=0.012), systolic BP (138+/-2 vs. 132+/-2, p=0.023), and diastolic BP (99+/-1 vs. 93+/-1, p=0.014) at baseline. To explore the molecular mechanisms of Caskin2’s effects, we used mass spectrometry to identify interacting proteins. Among the 67 proteins identified were the Ser/Thr phosphatase protein phosphatase 1 (PP1) and eNOS. Using standard in vitro biochemical techniques, we demonstrated that Caskin2 acts as a PP1 regulatory subunit. Interestingly, homologous expression of Caskin2 in vitro resulted in a marked increase in phosphorylation of eNOS on S1177, which is known to promote eNOS activity, and a decrease in phosphorylation on T495, which is associated with eNOS inhibition. Finally, PP1 has been shown to dephosphorylate eNOS T495 in vitro, suggesting a molecular mechanism for our in vivo findings. Ongoing work aims to determine if the interaction of Caskin2 and PP1 is required for the Caskin2-induced increase in activating phosphorylation of eNOS and to characterize the physiological mechanisms responsible for Caskin2’s effects on BP in more detail.
Abstract 069: Intrarenal Arteries Sense Mitochondrial N-formyl Peptides via Formyl Peptide Receptor in Wistar and Spontaneously Hypertensive Rats (shr)
Authors: Wenceslau Camilla F , McCarthy Cameron G , Ogbi Safia , O'Connor Paul M , Webb R. Clinton
Published: July 2021

It is well established that chronic immune system activation contributes to hypertension and kidney injury. Mitochondria carry hallmarks of their bacterial ancestry and thus have emerged as a significant source of inflammatogenic damage-associated molecular patterns (DAMPs). One of these hallmarks is that it still uses an N-formyl-methionyl-tRNA as an initiator of protein synthesis. Recently, we have observed that mitochondrial DAMPs are elevated in the circulation of SHR, and that mitochondrial N-formyl peptides (F-MIT) infusion in rats induces systemic inflammation and vascular dysfunction via formyl peptide receptor (FPR) activation. However, we do not know if FPR plays a role in kidney injury and hypertension. We hypothesized that F-MIT activate FPR and lead to intrarenal dysfunction in 12 week old male Wistar and SHR (n=4-8). Wistar rats were treated with F-MIT (0.02 mg/kg) or non-formylated peptide (control) for 6 h and intrarenal arteries (diameter >100 μm) were isolated. To exclude systemic effects of F-MIT, intrarenal arteries were also isolated from control rats and treated ex vivo with 100 nM F-MIT or non-formylated peptide. F-MIT treatment in vivo increased intrarenal arteries FPR protein expression (2.3-fold vs. control) and decreased β-arrestin 2 (protein that internalizes FPR upon activation) and phosphorylation of endothelial nitric oxide synthase (4-fold vs. control). These results were reproduced in isolated arteries incubated with F-MIT or control for 6 h ex vivo. Similarly, in intrarenal arteries from untreated SHR, we found that FPR protein expression was higher (1.5-fold vs. Wistar Kyoto, WKY) and β-arrestin 2 protein expression was decreased (2-fold vs. WKY). Interestingly, although treatment with hydrochlorothiazide (10-55 mg/kg/day) and reserpine (0.6-4.5 mg/kg/day) for 7 weeks attenuated the increase in blood pressure in SHR, anti-hypertensive therapy did not change FPR and β-arrestin 2 protein expression. Additionally, it was observed that the co-localization of FPR and β-arrestin 2 was decreased in intrarenal arteries from SHR. Overall, these data suggest that intrarenal arteries sense F-MIT. Also, FPR activation parameters following F-MIT treatment of normotensive rats are similar to those observed in SHR.
Abstract 076: Early Administration of 17-hydroxyprogesterone Caproate Improves Fetal Growth Restriction Possibly by Reducing Sflt-1 and Placental Cytolytic Nk Cells in Response to Placental Ischemia During Pregnancy
Authors: Amaral Lorena M , Elfarra Jamil , Cornelius Denise C , Faulkner Jessica L , Cunningham Mark W , Mcafee Taia R , Thomas D’Andrea S , LaMarca Babbette
Published: July 2021

Preeclampsia (PE), new onset hypertension, is characterized by elevated anti-angiogenic factor soluble fms-like tyrosine kinase (sFlt-1), cytolytic natural killer (NK) cells and placental ischemia predicted with increased uterine artery resistance (UARI) which are likely culprits for decreased fetal weight during PE pregnancies. Cytolytic NK are an important arm of the immune system killing tumor and infected cells by perforin-granzyme-mediated cytolysis and have been shown to be increased in PE women compared to those with normal pregnancy. Currently, there is no effective treatment for PE except for early delivery, making PE the leading cause for premature births worldwide. Administration of 17-hydroxyprogesterone caproate (17-OHPC) is used for prevention of spontaneous preterm labor, but is not included in the current management for PE. This study was designed to test the hypothesis that early administration of 17-OHPC could improve pregnancy outcomes in response to placental ischemia. To do so, 17-OHPC (3.32mg/kg) was administered intraperitoneally on gestation day 15 to reduced uterine perfusion pressure (RUPP) rats, UARI was measured using Doppler ultrasound and carotid catheters were inserted on day 18. Blood pressure (MAP), sFlt-1, and placental cytolytic NK cells were measured on GD 19. MAP in normal pregnant (NP) rats (n=12) was 94 ± 2, 126 ± 2 in RUPP (n=27) and 111±1 mmHg in RUPP+17-OHPC (n=15), p <0.05. Pup weight was 2.3±0.09 in NP, 1.9± 0.04 in RUPP rats, which improved to 2.1±0.06 grams in RUPP+17-OHPC p <0.05. UARI was 0.6±0.01 in NP (n=3), 0.8±0.03 in RUPP rats (n=4), which improved to 0.6±0.04 in RUPP+17-OHPC (n=5), p<0.05. Total number of placental NK cells was 8.6 ± 3.1 in NP, 20.2 ±2.4 in RUPP rats, which decreased to 1.6 ± 0.54 % in RUPP+17-OHPC, p<0.05. Activated placental NK cells was 3.8 ± 2.2 in NP, 11.9±2.01 in RUPP , which improved to 0.4 ± 0.2 % in RUPP+17-OHPC, p <0.05. Plasma sFlt-1 was 36.1±7.5, 385.9±141 in RUPP rats (n=5), which was blunted to 110.2±11.1 pg/mL in RUPP+17-OHPC, p<0.05. In conclusion, early administration of 17-OHPC improves sFtl-1, UARI, activated cytolytic NK cells, pup weight and hypertension in response to placental ischemia. Therefore, 17-OHPC should be further considered for addition to the management of PE.
Abstract 099: Fetuin-A and Toll-like Receptor 4 Regulate Vascular Function: Role of Nox1
Authors: Montezano Augusto C , Neves Karla B , Lopes Rheure A , Leckerman Susan , Strembitska Anastasiya , Jenkins Carol , Thomson Jacqueline , Graham Delyth , Touyz Rhian M
Published: July 2021

Fetuin-A (FetA) regulates calcium and phosphate homeostasis. It is also an agonist to toll-like receptor 4 (TLR4) and is related to insulin resistance and inflammation. FetA has also been associated with endothelial dysfunction, which is regulated by oxidative stress. Mechanisms whereby FetA influences vascular function are unknown. We hypothesized that FetA through TLR4 and ROS production induces vascular dysfunction. Mesenteric arteries and vascular cells from WKY rats were studied. Vascular function was analysed by wire myography in the presence or absence of FetA (50 ng/mL) and/or CLI095 (CLI - 10-6M - TLR4 inhibitor). Levels of reactive oxygen species (ROS) were measured by chemiluminescence, Amplex Red (H2O2) and ELISA (nitrotyrosine) Protein oxidation and levels were measured by immunoblotting. WKY vessels exposed to FetA were less sensitive to acetylcholine (Ach)-induced and sodium nitroprusside (SNP)-induced relaxation, while sensitivity to phenylephrine was increased by FetA; an effect blocked by N-acetylcysteine (antioxidant) and ML171 (Nox1 inhibitor). Inhibition of TLR4 blocked FetA effects on endothelial-dependent relaxation and contraction, but not on endothelial-independent relaxation. FetA increased ROS production (131±49.2%), but decreased H2O2 intracellular levels (63±14%) in endothelial cells (EC) (vs. veh, p<0.05); an effect blocked by CLI095. ROS production (66±12.2%), as well as, H2O2 (45±8%) and ONOO- (105±31.6%) levels, were increased by FetA in VSMCs (vs. veh, p<0.05). Protein oxidation was increased by FetA in VSMCs (103±26% vs. veh, p<0.05). In EC, eNOS inactivation (136±38%) and JNK activation (84±5%) were increased by FetA (vs. veh, p<0.05). In VSMCs, Rho kinase activity was increased (200±25% vs. veh, p<0.05) at 30 min; while myosin light chain (MLC) activation was only increased (25±3.56% vs. veh, p<0.05) at 15 min. In summary, FetA influences vascular function through Nox1-ROS dependent mechanisms. FetA-induced endothelial dysfunction and contractile responses involve TLR4. Our findings identify a novel system whereby FetA differentially influences vascular function through Nox1-ROS and TLR4. Vascular responses to FetA may depend on the specific pathway activated.
Abstract 109: Protective Effects of Nuclear Factor E2-related Factor 2 Activation on Angiotensin II Induced Microvascular Endothelial Dysfunction
Authors: Wang Cheng , Welch William J , Tomlinson James A , Leiper James , Wilcox Christopher S , Wang Dan
Published: July 2021

Background:
Nuclear factor E2-related factor 2 (Nrf2) is a regulator of the cellular adaptive response to oxidative stress, but its effects on microvascular function are poorly characterized. Our previous studies in human glomerular endothelial cells found that tert-butylhydroquinone (tBHQ; a Nrf2 activator) reduced ROS, asymmetric dimethylarginine (ADMA, an endogenous NOS inhibitor) and increased eNOS. We hypothesized that tBHQ would prevent microvascular endothelial dysfunction via activation of Nrf2 / antioxidant responses in angiotensin II (ANG II) induced hypertension.

Methods:
Mesenteric resistance arterioles (MRAs) were isolated from mice infused for 14 days with ANG II (400 ng/kg/min) or vehicle and given oral tBHQ (0.1% of water) or vehicle (n=6 mice/group). Acetylcholine-induced endothelium dependent relaxation (EDR), endothelial derived relaxation factor (EDRF) and contracting factor (EDCF) of MRAs were assessed by myograph. Vascular nitric oxide (NO), and ROS were assessed by RatioMaster. ROS biomarkers (urinary excretion of malondialdehyde [MDA]) and 8-isoprotane [8-Iso]), ADMA and protein expressions of MRAs were measured.

Results:
tBHQ prevented the effects (all P<0.05) of ANG II infusion-induced the increases of urinary MDA (50±3 vs 83±11 nmol/mg creatine) and 8-Iso (1.4±0.1 vs 3.8±0.3 vs ng/mg creatinine) and decreases of EDR (75 ± 3 vs 53±5%), EDRF (23± 2 vs 17± 3%) and NO (0.3 ± 0.02 vs 0.2 ± 0.03 units), and enhanced EDCF (7 ± 1 vs 13 ± 2%) and associated ROS production (0.11 ± 0.03 vs 0.3 ± 0.08 units) and microvascular ADMA (18 ± 1 vs 13 ± 1 nmol/mg protein). ANG II increased the MRAs protein expressions (of β-actin, all P<0.05) for NOX1 (0.60 ± 0.03 vs 0.38± 0.04) and NOX2 (0.80 ± 0.04 vs 0.36± 0.05) and reduced phosphorylated eNOS (0.26 ±0.07 vs 0.53±0.09), whereas tBHQ increased Nrf2 expression (0.44 ±0.03 vs 0.73±0.04) and prevented these changes with ANG II infusion.

Conclusions:
tBHQ upregulates Nrf2 and thereby engages the antioxidant responses to protect microvessels of ANG II-infused mice from the adverse effectors of ROS, NO deficiency and ADMA. This is a novel potential therapeutic target that activates Nrf2/antioxidant response element to protect against vascular oxidative stress, endothelial dysfunction and CVD.
Abstract 111: Maternal Separation, a Model of Early Life Stress in Rats, Dysregulates the Renal Vasculature Gene Expression Patterns During Late Nephrogenic Period and Adult Life
Authors: Chen Kuey-Chu , Loria Analia S
Published: July 2021

We have shown that maternal separation (MatSep) induces permanent alterations in the renal vascular structure, hemodynamics and sympathetic outflow. Environmental stressors are typically associated with abnormal organ development. Recently, we found that MatSep pups show higher levels of plasma corticosterone and renal norepinephrine during postnatal life (p< 0.05). The aim of this study was to investigate the short and long-term effects of MatSep on gene expression patterns of the renal vasculature. MatSep was performed during early postnatal life timeframe (3hr/day). Undisturbed littermates served as control (C). Upon weaning, rats were allowed to grow and develop for several months. Kidney vessels were isolated at postnatal day 10 (PND10, n=5) and 6 months of age (6Mo, n=5) and flash frozen. mRNA was used to perform genome-wide analysis using Affymetrix rat Gene2.0 ST. EBPfc* analysis was conducted using DAVID bioinformatics resources at NIH website. MatSep altered 1108 genes expression (P<0.03, FDR<0.3,Table 1). We found a set of genes upregulated by MatSep at both PND10 and 6Mo, showing long-lasting changes in expression. The uncoupling protein 1 (UCP1), located proximal to the major vascular and nerve conduits to the kidney, was the highest MatSep-induced upregulation (3.9-fold, PND10). UCP1 network of genes were mostly related to cellular organization and metabolic and cardiovascular disease (e.g. GH, AT1R, AT2R, P38 MAPK, IFNγR, VEGF, ApoE). Thus, MatSep influences timing and direction of key genes involved in the renal vascular maturation. Upregulated non-shivering thermogenesis function early in life may be linked to impaired renal function. NIH R00 HL111354
Abstract 117: NOS1β Mediates the Difference Between Juxtamedullary and Superficial Glomerular Injury with Aging
Authors: Wei Jin , Wang Shaohui , Zhang Jie , Jiang Kun , Yip Kay-Pong , Liu Ruisheng
Published: July 2021

Renal function measured by glomerular filtration rate (GFR) declines with aging. But the mechanism has not been clarified. Nitric oxide synthase 1 β (NOS1β) is highly expressed in the macula densa (MD) and modulates tubuloglomerular feedback (TGF), which is an important mechanism for GFR regulation. We hypothesized that expression of NOS1β in MD decreases with aging, which enhances TGF response and contributes to the decline of GFR. We further hypothesized that NOS1β expression in superficial (SF) MD is lower than that of juxtamedullary (JM) MD, which contribute to more severe renal injury in renal cortex with aging.

First, GFR was measured by plasma FITC-inulin clearance after a single bolus injection in conscious 12-week young mice and 20-month-old C57/BL6 mice. GFR was reduced by 36.4% in aged mice (236±9.7 vs 150±7.6 μl/min).

Then we compared the glomerular injury in 20-month-old C57/BL6 mice with 12-week mice. The aged mice have significant severed injury compared to the young mice as indicated by injury scores (3.6) and expanded fractional mesangial area (FMA) per glomerulus (26.4%). In addition, injuries were more evident in SF glomeruli with larger FMA (29.3%) than that in JM glomeruli (16.9%). Next we measured NOS1β protein abundance and found that NOS1β protein in aged mice was reduced by 73% compared to young mice. In addition, we compared the NOS1β abundance between SF and JM glomeruli in aged and young mice. NOS1β protein level decreased by more than 90% in SF nephrons in aged mice compared with the young mice. In contrast, there was no significant changes in NOS1β expression in JM nephron between aged and young mice.

To determine whether NOS1β mediates the changes in GFR and renal injury, we repeated the experiments in age matched MD specific NOS1β knock out (MD-NOS1KO) mice. In MD-NOS1KO mice, GFR in aged mice was reduced by 29.5% (203±15.7 vs 143±13.2 μl/min) compared young mice. Aged MD-NOS1KO mice had more severer renal injury compared with age-match wild type mice with higher injury score (4.7 vs 3.6), increased FMA (40.9% vs 26.4%) and occurrence of glomerulosclerosis (GS 30.8% ), but diminished difference between JM glomerulus (FMA37.4%, GS34.1%) and SF glomerulus (FMA42.2%, GS29.7%).
Abstract 122: Activation of Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) Enhances Cyclooxygenase 2 Expression via Promoter Antioxidant Response Element in Preglomerular Vascular Smooth Muscle Cells (PGVSMCs)
Authors: Luo Zaiming , Welch William J , Wilcox Christopher S
Published: July 2021

Nrf2 is a key cytoprotective transcription factor driving many antioxidant and anti-inflammatory genes expression via binding to antioxidant response elements (AREs) in promoters of targeting genes. However, it was reported that Nrf2 also increased expression of pro-inflammatory and pro-oxidative genes IL-6 and NOX4 through the same mechanism. We detected 2 putative ARE consensus sequences within 1500 nucleotides (nts) located in the genomic region upstream of the transcription start site in rat cyclooxygenase 2 (COX2) gene. Therefore, we hypothesized that activation of Nrf2-dependent AREs upregulates COX2 expression in rat PGVSMCs. Incubation of the cells for 24 hours with Nrf2 activator, tert-butylhydroquinone (tBHQ), did not significantly alter Nrf2 expression but dose-dependently increased COX2 gene and protein in a dose response manner (5-20μM). Twenty μM of tBHQ treatment enhanced expression of COX2 gene and protein by 10.7±2.2 and 9.2±3.4 fold (both p<0.01, N=3), COX2 activity (from COX2-based product of fluorescent resorufin) by 229.9±28.8% (p<0.005, N=6) but reactive oxygen species (ROS) generated in the cells (from oxidized dihydroethidium-based fluorescent intensity) were reduced by 35.1±10.2% (p<0.01, N=4). tBHQ also significantly promoted Nrf2 nuclear translocation by 86±7.4% (p<0.05, N=3) and potentiated Nrf2 activity by 387.5±19.2% (p<0.005, N=4) determined with cell fraction immunoblotting and Nrf2-ARE binding-based enzyme-linked immunosorbent assay. Nrf2 expression was dramatically knocked down with markedly decreased COX2 expression and activity in the cells transfected with Nrf2 specific siRNA and treated with tBHQ compared to those transfected with the scramble control siRNA and treated with tBHQ. Chromatin immunoprecipitation (CHIP)-based PCR analysis revealed an enhanced recruitment of Nrf2 to the endogenous COX2 promoter spanning the ARE (-417 to -426 nts) after 24 hour-tBHQ treatment but knockdown of Nrf2 gene with siRNA significantly diminished the tBHQ-mediated role, suggesting that there is a functional ARE located in COX2 promoter. In conclusion, these results show a novel role of Nrf2 in inducing COX2 expression through binding to promoter ARE in the absence of increased ROS in rat PGVSMCs.
Abstract 123: Age-Related Endothelial Senescence is Driven by Thrombospondin-1-Activated NADPH Oxidase Nox1
Authors: Meijles Daniel N , Al Ghouleh Imad , Sahoo Sanghamitra , Amaral Jefferson H , Knupp Heather , Attaran Shireen , Bienes-Martinez Raquel , Sembrat John C , Rojas Mauricio , Isenberg Jeffrey S , Pagano Patrick J
Published: July 2021

Organismal aging represents an independent risk factor underlying many vascular diseases, including systemic and pulmonary hypertension, and atherosclerosis. While the mechanisms driving aging are largely elusive, a steady persistent increase in tissue oxidative stress has been associated with senescence. Previously we showed TSP1 elicits NADPH oxidase (Nox)-dependent vascular smooth muscle cell oxidative stress. However mechanisms by which TSP1 affects endothelial redox biology are unknown. Here, we tested the hypothesis that TSP1 induces endothelial oxidative stress-linked senescence in aging. Using rapid autopsy disease-free human pulmonary (PA) artery, we identified a significant positive correlation between age, protein levels of TSP1, Nox1 and the cell-cycle repressor p21cip (p<0.05). Age also positively associated with increased Amplex Red-detected PA hydrogen peroxide levels (p<0.05). Moreover, treatment of human PA endothelial cells (HPAEC) with TSP1 (2.2nM; 24h) increased expression (~1.9 fold; p<0.05) and activation of Nox1 (~1.7 fold; p<0.05) compared to control, as assessed by Western blot and SOD-inhibitable cytochrome c reduction. Western blotting and immunofluorescence showed a TSP1-mediated increase in p53 activation, indicative of the DNA damage response. Moreover, TSP1 significantly increased HPAEC senescence in a p53/p21cip/Rb-dependent manner, as assessed by immunofluorescent detection of subcellular localization and senescence-associated β-galactosidase staining. To explore this pathway in vivo, middle-aged (8-10 month) wild-type and TSP1-null mice were utilized. In the TSP1-null, reduced lung senescence, oxidative stress, Nox1 levels and p21cip expression were observed compared to wild-type supporting findings in human samples and cell experiments. Finally, prophylactic treatment with specific Nox1 inhibitor NoxA1ds (10μM) attenuated TSP1-induced HPAEC ROS, p53 activation, p21cip expression and senescence. Taken together, our results provide molecular insight into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence, with implications for molecular control of the aging process.
Abstract 129: A Biopolymer-Stabilized VEGF Chimera Reverses Angiogenic Imbalance
in vitro
in the Reduced Uterine Perfusion Pressure Model of Preeclampsia
Authors: Logue Omar C , Mahdi Fakhri , George Eric M , Bidwell Gene L
Published: July 2021

Preeclampsia (PE) is a hypertensive disorder that complicates approximately 5-8% of all pregnancies in the U.S. The unknown etiology of PE develops from molecular dysfunction at the maternal-placental interface, where inflammatory, oxidative stress, and anti-angiogenic pathways are initiated. The resulting hypoxic
in utero
environment contributes to placental ischemia from which the anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1) is released. A truncated isoform, sFlt-1 consists solely of the extracellular domain of the vascular endothelial growth factor (VEGF) receptor 1, and hence lacks both catalytic and regulatory activities. However, sFlt-1 retains a high affinity for VEGF and sequesters this ligand from binding with fully functional VEGF receptors, thus prevents the activation of angiogenic remodeling pathways. The rodent model of PE, reduced uterine perfusion pressure (RUPP), exhibits the sFlt-1 pathophysiology observed in human PE. In an effort to counteract excessive sFlt-1 production and restore angiogenic balance, we have constructed a VEGF chimera fused to an Elastin-like Polypeptide (ELP) carrier with increased in vivo half-life and stability which retains full VEGF signaling activity. When human umbilical vein vascular endothelial cells (HUVECs) are exposed to serum from normal pregnant or RUPP-treated rats, tube formation on extracellular matrix is inhibited 31% (± 2%) by the RUPP serum. This inhibition is reversed when ELP-VEGF, but not ELP control, is added to the culture medium (
p
= 0.0007, one-way ANOVA with Bonferroni multiple comparison), suggesting that ELP-VEGF counteracts the anti-angiogenic factors present in RUPP serum. We also characterized the pharmacokinetics, biodistribution, and placental transfer of ELP-VEGF in the pregnant Sprague Dawley rat. These

studies indicate that ELP-delivered VEGF has potential for counteracting the circulating anti-angiogenic factors in maternal plasma.
Abstract 132: Leptin-mediated Aldosterone Secretion Causes Hypertension in Obese Females
Authors: Belin de Chantemèle Eric J , Cortez-Cooper Miriam , Cannon Joseph , Huby Anne-Cécile
Published: July 2021

Obesity causes hypertension (HTN) in males and females. While leptin contributes to obesity-induced HTN by increasing sympathetic activity, in males, it is unknown whether similar mechanisms trigger HTN in obese females. Females secrete 3 to 4 times more leptin than males, but do not exhibit high sympathetic tone with obesity. They however show inappropriately high aldosterone levels that positively correlate with adiposity and blood pressure (BP). Here we hypothesized that leptin induces HTN by increasing aldosterone production in obese females. Hypersensitivity to leptin, in lean mice deficient in protein tyrosine phosphatase 1B (PTP1B) or high leptin levels, in obese Agouti (Ay/a) mice induced HTN (WT: 115±2; KO: 124±2; a/a: 113±1; Ay/a: 128±7mmHg, p<0.05) but did not increase sympathetic control of BP (response to ganglionic blockade). Leptin sensitization and obesity however elevated plasma aldosterone levels and adrenal aldosterone synthase (CYP11B2) expression, in females. Chronic leptin (KO+AA: 115±5; Ay/a+AA: 114±5mmHg) or mineralocorticoid (KO+spiro:111±5; Ay/a+spiro: 121±6mmHg) receptors inhibition restored BP to baseline levels in females PTP1B KO and obese agouti mice. Leptin or leptin receptor deficiency in female ob/ob and db/db mice, abolished obesity-induced increases in adrenal CYP11B2 and plasma aldosterone while chronic leptin infusion in female mice triggered a dose-dependent increase in adrenal CYP11B2 and plasma aldosterone levels. Leptin-mediated aldosterone secretion was independent of changes in plasma angiotensin II, potassium and corticosterone (index of ACTH levels) and preserved in the presence of losartan or α and β-adrenergic receptors antagonists. Stimulation of human adrenocortical cells with leptin dose-dependently increased CYP11B2 expression and aldosterone production. While investigating the interaction between percentage of body fat, leptin and aldosterone levels in young healthy adult Caucasians we reported a positive correlation between adiposity and aldosterone, and between leptin and aldosterone in adult women only. Together these data suggest that leptin directly regulates aldosterone secretion and that leptin induces HTN via aldosterone dependent mechanisms in obese females.
Abstract 133: Role of Vascular and Myeloid Mineralocorticoid Receptor in Renal Ischemia/reperfusion
Authors: Barrera-Chimal Jonatan , Le Mercier Alan , Prince Sonia , Fadel Fouad , El Moghrabi Soumaya , Bobadilla Norma A , Jaisser Frederic
Published: July 2021

Introduction:
Renal ischemia/reperfusion (IR) is a major cause of acute kidney injury. It is associated with cardiac alterations and chronic kidney disease (CKD) development. We previously showed that mineralocorticoid receptor (MR) antagonism prevents the acute and chronic consequences of renal IR (Barrera-Chimal, Kidney Int, 2013 and JASN, 2015). However, whether the benefit of the MR antagonists is due to the blockade of the MR expressed in the vessels is unclear.

Objective:
To study the specific contribution of endothelial and smooth muscle cells MR in acute and chronic consequences of renal IR.

Methods:
To evaluate the contribution of vascular MR we generated two knockout (KO) mouse models. To allow MR inactivation in endothelial cells (MR
endoKO
mice), floxed MR mice (MR
fl/fl
) were crossed with mice expressing the inducible Cre recombinase under the VEcadh promoter. To allow MR inactivation in vascular smooth muscle cells (MR
SMCKO
mice), MR
fl/fl
mice were crossed with mice expressing the inducible Cre recombinase under the SMA promoter. In these mice, sham surgery or bilateral renal IR for 20 min was performed in MR
fl/fl
and KO mice and the animals were studied at short term (24 h) and long term (30 days) after reperfusion.

Results:
In MR
fl/fl
mice, IR induced renal dysfunction (plasma creatinine raised from 8.9±0.3 in sham to 33.8±4.8 umol/L in IR), tubular injury and increased mRNA levels of kim-1 (400-fold) and NGAL (220-fold). The MR
endoKO
mice displayed similar alterations induced by IR as MR
fl/fl
mice. In contrast, after 24 h of renal IR, the MR
SMCKO
mice presented normal renal function (plasma creatinine was 9.6±0.7 and 14.0±1.9 umol/L in sham and IR, respectively), absence of histological alterations and reduced kim-1 and NGAL levels.

After 30 days, the MR
fl/fl
mice developed CKD characterized by renal dysfunction (plasma creatinine from 10.5±0.1in sham to 15±0.8 umol/L in IR), tubule-interstitial fibrosis and increased mRNA levels of fibronectin and Galectin-3 (2-fold). The MR
SMCKO
mice developed similar alterations.

Conclusion:
We provide evidence that the deficiency of MR in the SMC protects against the development of acute kidney lesions induced by IR, however MR deficiency in SMC did not impact the appearance of CKD induced by IR.
Abstract P004: Telemonitoring of Blood Pressure in Low-Income African American Patients With Congestive Heart Failure
Authors: Shah Gautam V , Reali-Sorrell Michele , Barzilai Benico , Brateanu Andrei
Published: July 2021

Introduction:
Uncontrolled blood pressure (BP) due to non-adherence to medical therapy or inadequate medical therapy in African American (AA) patients with congestive heart failure (CHF) increases the risk of CHF exacerbation. Use of telemonitoring (TM) to monitor BP in low-income AA patients with CHF has not been studied.

Aim:
To identify patients with uncontrolled BP in low-income AA patients with CHF using a TM device.

Methods:
We conducted a prospective study of 15 patients with CHF (Ejection Fraction <45%) randomly selected from a Cleveland Clinic outpatient clinic serving a low income AA population. Patients were trained to use a tablet monitor & a BP machine at their home to record their BP everyday for a period of 28 days. Using a secure internet connection patients BP measurement was transmitted to a password protected website where it was studied daily by a nurse to identify abnormal parameters (BP equal to or greater than 140/90 mm of Hg). The nurse contacted patients with abnormal BP measurements to ensure medication adherence.

Results:
15 patients consented to participate in the study. During the study period 67% (10/15) patients used the TM device to record BP for more than 50% (14/28 days) of the days. Of these, 50% (5/10) patients had either systolic or diastolic blood pressure equal to or greater than 140/90 mm of Hg for more than 50% of the times they were monitored (Figure 1).

Conclusions:
Use of TM can help identify patients with CHF whose blood pressure is not at goal and may increase patient’s medication adherence. Whether TM is a cost effective tool to improve clinical outcomes in low income AA patients with CHF & uncontrolled BP needs to be proven in larger studies.
Abstract P006: Liraglutide Attenuates Cardiac Fibrosis and Vascular Dysfunction in a Non-diabetic Angiotensin II-infusion Model via Anti-inflammatory and Anti-oxidant Mechanisms
Authors: Lee Huey Wen , Brdar Melita , Widdop Robert , Dear Anthony , Gaspari Tracey
Published: July 2021

Glucagon-like peptide-1 (GLP-1) based therapies are used to treat type II diabetes via increasing insulin secretion and inhibiting glucagon production. Recent evidence suggests that activating the GLP-1 receptor may also mediate direct vaso-protective effects. Therefore the objective of the study was to determine whether GLP-1R stimulation conferred cardio- and vaso-protection in a non-diabetic setting using the angiotensin (Ang) II infusion model of hypertension and cardiovascular dysfunction. Male C57Bl/6J mice (4-6 months) were assigned to one of the following 4 week treatment protocols: 1) vehicle (saline), 2) Ang II (800ng/kg/day), 3) Ang II + liraglutide (30μg/kg/day), 4) Ang II + liraglutide (300μg/kg/day). All treatments were administered via osmotic mini-pumps (s.c). After 4 weeks the effect of liraglutide treatment on blood pressure, vascular function and cardiac remodelling was examined. Liraglutide (both doses) attenuated Ang II-induced increase in systolic blood pressure (Ang II: 175.3 ± 8.6mmHg vs Ang II+Lirag (30) 150.2 ± 6.4 mmHg or Ang II+Lirag (300): 145.4 ± 6.9 mmHg) without affecting blood glucose levels. Liraglutide (both doses) completely prevented Ang II-induced endothelial dysfunction (% maximum relaxation: Ang II=50.7 ± 7.8%; Ang II+Lirag (30)=82.7 ± 5.8; Ang II+Lirag (300)=81.5 ± 6.1%). In the heart, liraglutide prevented Ang II-induced cardiomyocyte hypertrophy (n=7-10; p<0.05) and reduced collagen deposition (% collagen expression: Ang II=4.4 ± 0.5 vs Ang II+Lirag(300)=2.9 ± 0.3; n=7-9; p<0.01). This anti-fibrotic effect was attributed to reduced fibroblast/myofibroblast expression as well as decreased inflammation with reduced NFκB and MCP-1 expression and decreased oxidative stress with a significant reduction in superoxide production using high dose of liraglutide. Overall, stimulation of GLP-1R in a non-diabetic setting protected against Ang II-mediated cardiac hypertrophy, cardiac fibrosis and vascular dysfunction, indicating potential for use of GLP-1 based therapies in treatment of cardiovascular disease independent of diabetes.
Abstract P010: Specific Renal Outer Medulla Upregulation of α-and β-Adrenergic Receptor Subtypes in Spontaneously Hypertensive African Green Monkeys
Authors: Rhoads Megan K , Osborn Jeffrey L
Published: July 2021

The African Green Monkey (
Chlorocebus aethiops sabaeus
) is a highly translational model of spontaneous hypertension (HT). Within the cohort of measured colony animals, 33% (98 of 300) of adult African Green Monkeys exhibit systolic blood pressures (SBP)> 140 mmHg, assessed by forearm cuff plethysmography. Heart rate is elevated in HT vs. normotensive (NT) animals (121±2 bpm vs. 134±2 bpm, p<0.05) suggesting that sympathetic nerve activity is likely elevated in the HT African Green Monkey, similar to patients with essential hypertension. This study assessed mRNA expression of adrenergic receptor subtypes in the renal cortex, outer medulla, and inner medulla of male HT and NT African Green Monkeys. Primers were custom designed and evaluated for appropriate specificities and efficiencies (95-105%) for α
1a
, α
1d
, α
2c
, β
1
and β
2
adrenoceptors. Gene expression was measured with qRT-PCR and normalized to RPS32 expression in the liver of each animal. The HT cohort of African Green Monkeys (n=18) assessed an averaged SBP of 168.24 ± 7.25mmHg compared with NT animals (n=18) that averaged 96.61 ± 3.20mmHg. Expression of α
1a
, α
1d
and α
2c
genes were all increased in the renal outer medulla of HT animals (α
1a
: NT RQ 0.24 ± 0.15 vs. HT RQ 0.89 ± 0.22, p<0.05; α
1d
: NT RQ 0.43 ± 0.24 vs. HT RQ 1.99 ± 0.74, p<0.05; and α
2c
: NT RQ 1.11 ± 0.53 vs HT RQ 1.41 ± 0.35, p<0.05). β
1
adrenergic receptor expression in the outer medulla was similar in NT and HT animals (β
1
: NT RQ 0.75 ± 0.30 vs. HT RQ 1.82 ± 0.64, p>0.05), but β
2
adrenoceptor expression was upregulated in HT animals (β
2
: NT RQ 0.36 ± 0.13 vs HT RQ 2.05 ± 0.82; p<0.05). Expression of each adrenoceptor subtype was similar in the renal cortex and inner medulla of HT animals (p>0.05, all groups compared with Mann-Whitney U Test). These data suggest that the renal outer medulla of HT African Green Monkey may be sensitized to sympathetic nerve activity via elevated adrenergic receptor gene expression. Thus, subtle changes in renal sympathetic outflow may elicit renal medullary vasoconstriction and contribute to the development of the spontaneous hypertensive phenotype in the African Green Monkey.
Abstract P019: Upregulation of Autophagy Enhances (pro)renin Receptor Expression via P62 -erk1/2 Signaling Pathway in Podocytes
Authors: Li Caixia , Quadri Syed , Siragy Helmy M
Published: July 2021

Abnormally up-regulated or down-regulated autophagy induces cell death. p62 is selectively degraded by autophagy, and was also recently reported to modulate extracellular signal-regulated kinases (ERK1/2) activity. However, the effect of autophagy on PRR expression is not known. We hypothesized that autophagy activation increases PRR expression via modulation of p62 -ERK1/2 signaling pathway. Cultured mouse podocytes were treated with the autophagy activators, rapamycin (100 nm) or Earles’s balanced salt solution (EBSS), for 48 hours. Both rapamycin and EBSS significantly increased mRNA and protein expressions of PRR (see below attached figures), decreased p62 protein levels and increased ERK1/2 activation by phosphorylating pTpY185/187. Confocal microscopy studies showed significant increase in protein levels of microtubule-associated protein-1 light chain 3B (LC3B) and PRR, and decrease of p62/SQSTM1 level in response to rapamycin or EBSS. Using autophagy related 5 (ATG5) cDNA or ATG7 cDNA transfection for 72 hours to enhance podocytes autophagy showed the same results as rapamycin and EBSS treatments. Inhibition of autophagy with bafilomycin A1 (10 nm) reversed the effects of rapamycin. ERK1/2 inhibitor U0126 (10 μM) significantly attenuated mRNA and protein expressions of PRR in podocytes treated with rapamycin, while has no effect on p62/SQSTM1 level. In conclusion, up-regulation of autophagy enhanced PRR expression through p62-ERK1/2 signaling pathway.
Abstract P024: Pharmacological Treatment of Hypertension and Lv Dysfunction Predictors in ESRD Patients With AV Fistula
Authors: Tariq Sayed , Anderson James , Dhingra Rohit , Torosoff Mikhail
Published: July 2021

Background:
Effects of anti-hypertensive medications on left ventricular dimensions and systolic function in patients with arterio-venous (AV) fistulas have not been well investigated.

Material and Methods:
Medical charts and echocardiograms of 346 patients with AV fistula were reviewed. Of 346, 149 patients had TTE prior to the AV fistula surgery, 197 had TTE after the AV fistula surgery, and 76 patients had TTE before and after the AV fistula surgery. Data on medication use was available in 314 patients. ANOVA, chi-square, and logistic regression tests were employed.

Results:
In patients scheduled for AV fistula placement, 20% (31/149) patients had systolic dysfunction and 15% (22/142) had increased LV end-diastolic dimensions (LVEDD). Moderate systolic LV dysfunction was observed in 6% (9/149) and additional 8% (12/149) had severe LV dysfunction. Increased LVEDD with some LV dysfunction was noted in 27% (38/142).Following the AV fistula placement, 18% (36/197) of patients had systolic dysfunction and 12% (22/187) had increased LV end-diastolic dimensions (LVEDD). Moderate or severe systolic LV dysfunction was observed in 6% (5/197). LV systolic dysfunction or dilatation was noted in 23% (43/187).

Of 314 patients, 63% were on beta-blockers (BB), 25% were on ACE inhibitor or an ARB , 43% on calcium-channel blocker , and 15% on alpha-antagonist . BB, ACEi-ARB, or AA were not associated with increased LVEDD or systolic dysfunction before or after the AV fistula placement. Prior to AV fistula, CCB treatment was not related to LV dilatation (36% in each group, p=0.981)
Post AV fistula, CCB treatment was associated with increased LV dimensions (71% vs. 46%, p=0.029) but not LV systolic dysfunction (49% in LV dysfunction vs. 38% in the rest, p=0.446)
. This association persisted after adjustment for co-morbidities and demographic parameters.

Conclusions:
LV systolic dysfunction and/or dilatation are common in patients undergoing AV fistula surgery. Despite decreased use of Ca-channel blockers in patients with LV dysfunction prior to AV fistula, Ca-channel blockers are associated with increased LV dimensions post AV fistula, and probably should be avoided in this vulnerable patient population.
Abstract P034: Endothelin-1 Overexpression Exaggerates Diabetes-induced Endothelial Dysfunction by Altering Oxidative Stress Balance
Authors: Idris Khodja Noureddine , Ouerd Sofiane , Mian Muhammad Oneeb Rehman , Gornitsky Jordan , Barhoumi Tlili , Paradis Pierre , Schiffrin Ernesto
Published: July 2021

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

Method:
Diabetes was induced by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days in 6weekold male wild-type (WT) mice and in mice overexpressinghuman ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Blood glucose,plasma ET-1 levels, mesenteric artery (MA) reactivity, mitochondrial superoxide production in aorta and endothelial nitric oxide synthase (Nos3), superoxide dismutase 1 (Sod1) and 2 (Sod2) mRNA expression in MA were determined.

Results:
STZ-induced diabetes was confirmed by increased glycemia in WT(27.6±1.5 vs 10.7±1.1 μM, P<0.001)and eET-1 (23.2±1.0 vs 8.4±0.3 μM, P<0.001).Plasma ET-1 was increased in vehicle- (15.9±4.6 vs 0.6±0.04pg/mL, P<0.05) and STZ-treatedeET-1 (4.9±0.6 vs 0.8±0.1 pg/mL, P<0.05) compared to respective WT controls.Diabetes caused a reduction in vasodilatory responses to acetylcholine in WT (60.9±6.4% vs 83.9±3.4%,P<0.05), which was exaggerated in eET-1(48.6±5.1% vs 81.5±5.2% P<0.05). Mitochondrial superoxide production was increased by diabetes in WT (38.0±4.3 vs 21.6±2.3 RFU/μm2,P<0.05)and further augmented in eET-1 (49.8±1.7 RFU/μm2P<0.05).Nos3 expression was increased in vehicle-treatedeET-1 compared to WT(1.43±0.19 vs 1.00±0.10, P<0.05).Diabetes reduced Nos3 expression in eET-1 (0.75±0.08, P<0.05) but not in WT(1.08±0.09).Diabetes caused an increase in Sod1(1.52±0.17 vs 1.00±0.03, P<0.05) and Sod2 (1.32±0.17 vs 1.00±0.02, P<0.05) expression in WT (P<0.05) but not in eET-1.

Conclusions:
Increased levels of ET-1 exaggerate diabetes-induced endothelial dysfunction. This may be caused by a decrease in Nos3 expression, an increase in mitochondrial oxidative stress and a decrease in antioxidant capacity.