bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 010: CD44 and CD44+ Cells are Dispensable for the Recruitment of Renin Expressing Cells
Authors: Sequeira Lopez Maria Luisa S , Belyea Brian C , Kaur Rajwinderjit , Medrano Silvia , Gomez R. Ariel
Published: June 2021

In response to a homeostatic stress the number of cells that make renin increases dramatically along the renal arteriolar tree resembling the embryonic pattern. We have shown that this “recruitment” occurs by re-expression of renin in smooth muscle cells that differentiated from embryonic renin cells. A recent study proposed that during recruitment, renal CD44+ mesenchymal stem-like cells can differentiate into juxtaglomerular (JG)-like renin-producing cells. To test such hypothesis, we assessed the distribution and role of CD44+ cells in renin cell recruitment. Mice with homozygous (KO) and heterozygous (het) deletion of CD44 (knockin for LacZ) were treated with low-sodium diet (0.05%) plus captopril (0.5 g/l) for 10 days (n: 9 treated, 7 controls). Body and kidney weights and BP were not different between KO and het mice. BUN and creatinine were significantly increased in both KO and Het treated mice. The number of renin expressing cells in the kidney and circulating renin increased similarly in treated mice (ELISA, untreated: het 131,503 ± 19,319 pg/mL vs KO 84,714 ± 29,065 pg/mL p=0.2517; treated: het 367,850 ± 38,189 pg/mL vs KO 495,120 ± 80,311 pg/mL p=0.2311). Interestingly, immunostaining for CD44 was negative in kidneys of untreated and treated wild type mice. We occasionally observed in CD44-LacZ het or KO mice isolated LacZ positive cells inside the glomeruli (1 or less per sagittal kidney section) and none in the JG area. On the other hand, immunostaining for CD44 on kidney sections of Ren1cKO mice revealed positive cells within perivascular infiltrates. To confirm these results we performed qRT-PCR for CD44 on kidney samples from CD44 het and KO treated, untreated, control, and Ren1cKO mice. CD44 mRNA expression confirmed the histological findings. In summary: 1) CD44 is dispensable for renin expression and recruitment, and 2) CD44+ cells do not contribute to the pool of renin expressing cells in the kidney during basal conditions or in response to a homeostatic stress as previously suggested. However, they do participate in the inflammatory process observed surrounding the vessels in mice with deletion of the renin gene, suggesting that they derived from the circulation and not from the kidney.
Abstract 013: Genetic Ablation of Mas Receptor Impairs Mobilization of Bone Marrow Progenitor Cells
Authors: Vasam Goutham , Joshi Shrinidh , Thatcher Sean , Cassis Lisa A , Jarajapu Yagna P
Published: June 2021

Angiotensin (Ang)-(1-7)/Mas receptor (MasR) pathway accelerates vascular repair in ischemic conditions partly by stimulating the mobilization of vascular reparative bone marrow progenitor cells (BMPCs) into blood circulation. This study tested if the endogenous MasR expression is required for the mobilization of BMPCs in response to ischemic injury. Hind limb ischemia (HLI) was induced in wild type (WT) or MasR knock out mice (MasR-KO) (in C57Bl/6J background). BMPCs in the blood circulation were quantitated by flow cytometric enumeration of Lineage
-
, Sca-1
+
and cKit
+
(LSK) cells in peripheral blood or by colony forming unit (CFU) assay. Subcutaneous osmotic pumps were used for continuous infusion of Ang-(1-7) at the rate of 1 μg/kg/min for four weeks. In vitro migration of LSK cells in response to hypoxia-regulated factors, stromal-derived factor (SDF) or by vascular endothelial growth factor (VEGF) were determined. In WT mice, HLI stimulated mobilization of LSK cells that reached maximum by day 2 (110±11 cells/mL blood, n=6). Ang-(1-7)-treatment potentiated the peak mobilization (206±24 cells/mL blood, n=8, P<0.01 compared to the untreated). MasR-KO mice have reduced number of circulating LSKs (12±3 vs 43±9 per mL blood in WT, P<0.01, n=5) (CFUs/mL blood 28±5 vs 54±8 in WT, P<0.05, n=5). In MasR-KO mice, HLI did not induce mobilization, and blood flow recovery post-HLI was lower compared to WT (52±4% vs 89±6% in WT, P<0.001, n=5), both of which were not improved by treatment with Ang-(1-7). Number of bone marrow-resident LSK cells was higher in MasR-KO mice compared to WT. Migration induced by SDF (84±6% vs 160±8% in WT, P<0.001, n=5) or VEGF (97±4% vs 146±5% in WT, P<0.001, n=4) was decreased in MasR-KO. These results suggest that MasR deficiency causes impaired mobilization of BMPCs likely by decreasing their sensitivity to hypoxia-regulated factors. Therefore endogenous MasR expression is essential for ischemia-dependent mobilization of BMPCs.
Abstract 016: Cells Programmed for the Renin Phenotype Contribute to Vascular Pathology in Renin Deficient Mice
Authors: Oka Masafumi , Medrano Silvia , Sequeira-Lopez Maria Luisa S , Gomez R A
Published: June 2021

Deletions of the renin-angiotensin system genes or pharmacological inhibition in early life result in a distinctive renal pathology: concentric and disorganized intra-renal arteriolar thickening. The origin and distribution of the cells contributing to the arterial disease are not known. Because the arteriolar thickening disappears with ablation of renin cells, we hypothesized that renin cell precursors contribute to the arterial pathology. To reveal the origin and distribution of the cells responsible for the arterial thickening we generated several mouse lines for fate tracing and also stained for cell identity specific proteins. Kidneys from
Ren1c-/-
(n=6) and
Ren1c+/-
(n=6) mice were immunostained for renin, αSMA and PECAM1. Arterial wall thickness was measured using a light microscope and the Leica MM AF
®
version1.5 software. Renin cells (unable to produce renin because of the knock out) were identified using
Ren1c-/-; Ren1c-YFP
mice, where the yellow fluorescent protein is expressed by the
Ren1c-YFP
transgene designed to label all cells with an active renin promoter. In addition, we tracked the expression and distribution of aldo-keto reductase 1b7, AKR1b7, which mark cells programmed for the renin phenotype even when renin is absent. As expected,
Ren1c-/-
kidneys showed no renin and thicker intra-renal arteries (Arterioles:
Ren1c+/-
, 8.26 ± 2.5 μm
vs.
Ren1c-/-
, 14.3 ± 3.8 μm,
P<0.0001
, larger arteries:
Ren1c+/-
, 29.2 ± 11.1 μm
vs.
Ren1c-/-
, 42.1 ± 11.1 μm,
P<0.0001
) AKR1b7+ and YFP+ cells were retained and observed throughout the renal arterioles. To investigate the fate and distribution of cells from the renin lineage, we used
Ren1c-Cre
and
R26R.LacZ
or
mT/mG
reporter mice (6 knock out and 6 control mice per strain). Cells from the renin lineage surrounded arterioles and persisted within larger arterial walls whereas PECAM1+ endothelial cells did not contribute to the arterial wall thickening. In control mice, renin cells were confined to the juxtaglomerular area. We conclude that precursor cells programmed for the renin phenotype maintain their molecular program and together with vascular smooth muscle cells contribute to nephro-vascular disease.
Abstract 025: AMP-activated Protein Kinase Mediates Pressure Overload-induced Hypertrophy
Authors: Wang Jinli , Quan Nanhu , Wang Lin , Cates Courtney , Li Ji
Published: June 2021

Hypertension is a major risk factor of death and disability from heart and vascular diseases. Heart hypertrophy caused by pressure overload is characterized by the activation of adenosine monophosphate-activated protein kinase (AMPK), which is the major energy sensor in the heart. However, the anti-inflammatory effect of AMPK has not been investigated in the hypertrophy model. Activated protein C (APC) is a vitamin-K dependent plasma serine protease which inhibits blood clotting, and APC is an endogenous AMPK agonist. This study was designed to examine the role of AMPK in hypertrophy and the underlying mechanisms by which APC inhibits heart hypotrophy by pressure overload. We hypothesize that AMPK play a role in preventing heart from hypertrophy induced by pressure overload. APC could inhibit high blood pressure-induced hypertrophy via activation of AMPK signaling pathway. Wild-type (WT) and AMPK-kinase dead (KD) transgenic mice were subjected to transverse aortic constriction (TAC) surgery. Echocardiography was performed to evaluate the heart function, and histology staining revealed the morphological changes. Real-time PCR and western blotting were used to detect the signaling changes of both mRNA and protein expression levels. There is no phenotype difference between WT and AMPK-KD mice under normal physiological conditions. However after 4 weeks of TAC surgery, AMPK-KD mice demonstrated significantly bigger heart than WT mice (p<0.05), and the cardiac functions measured by echocardiography in AMPK-KD hearts were significantly impaired as compared with WT hearts (p<0.05). The immunohistochemical staining showed that the increased macrophage infiltration and reactive oxygen species (ROS) including activated p66shc, 4-HNE and ERK were observed in the AMPK-KD hearts after 4 weeks of TAC surgery (all p<0.05 versus WT hearts). APC administration significantly attenuated hypertrophy and fibrosis caused by pressure overload, and macrophage infiltration and p66shc activation were also inhibited by APC treatment. Therefore, Cardiac AMPK deficiency aggravates hypertrophy caused by pressure overload. AMPK activator APC could be a therapeutic drug for treatment of hypertrophy by high blood pressure.
Abstract 030: Low-dose Aspirin During Pregnancy Does Not Prevent Augmentation of Arterial Contractions to Thromboxane A
2
in a Rat Model of Pregnancy-Induced Hypertension
Authors: Osikoya Oluwatobiloba , Jaini Paresh A , Nguyen An , Valdes Melissa , Goulopoulou Styliani
Published: June 2021

Background:
Daily intake of low dose aspirin after 12 weeks of gestation is currently recommended as a preventative intervention in pregnancies at high risk of developing preeclampsia. We previously showed that treating pregnant rats with a synthetic ligand of Toll-like receptor 9 (TLR9; ODN2395) activated the innate immune system causing preeclampsia-like characteristics such as maternal hypertension, vascular oxidative stress, and augmented vascular responses to thromboxane A
2
(TxA
2
).

Hypothesis:
Low-dose aspirin treatment would ameliorate ODN2395-induced augmented responses to TxA
2
in maternal arteries.

Methods:
Pregnant rats were treated with ODN2395 (300 μg) or vehicle on gestational day (GD) 14, 16, 18. Daily low-dose aspirin treatment (1.5 mg/kgBW) started on GD10 and continued throughout gestation. Systolic blood pressure was measured using the tail-cuff method on GD19. On GD21, uterine (UTA) and mesenteric resistance artery (MES) responses to a TxA
2
mimetic (U46619, 10
-9
-10
-6
M) was measured with a wire myograph.

Results:
Uterine arteries from the ODN2395+Aspirin-treated group had greater contractile responses to U46619 compared to all other groups [Emax, %KCl: Control (n=6): 109.2±8.8; ODN2395 (n=5): 124.4±20.2; Aspirin (n=5): 76.08±11.2; ODN2395+Aspirin (n=5): 134.7±4.5, p<0.05]. Aspirin alone reduced MES responses to U46619 compared to control and MES from ODN2395 and ODN2395+Aspirin groups had greater contractile responses to U46619 compared to Aspirin alone [Emax, %KCl: Control (n=8): 109.8±5.2; ODN2395 (n=6): 111.1±4.3; Aspirin (n=7): 89.4±3.6; ODN2395+Aspirin (n=8): 121.7±6.3, p<0.05]. Rats treated with ODN2395 and ODN+Aspirin had greater systolic blood pressure compared to control rats [control (n=8): 97.3±3.2 mmHg, ODN2395 (n=6): 121.3±4.3 mmHg, Aspirin (n=8): 100.3±2.9 mmHg, ODN2395+Aspirin (n=7): 127.5 ±6.7 mmHg, p<0.05].

Conclusion:
Exposure to a TLR9 agonist during pregnancy resulted in augmented contractile responses to a TxA
2
mimetic in maternal arteries when rats were in a regime of low-dose aspirin. This was shown in arteries from both reproductive and non-reproductive vascular beds.
Abstract 034: Characterization of A Novel Maternally Restricted Pro-angiogenic Therapeutic for Preeclampsia
Authors: Stewart William H , George Eric , Bidwell Gene L , Chapman Heather , Mahdi Fakhri , Wilson Logan
Published: June 2021

Background:
Preeclampsia is a major obstetrical health concern, affecting 5-8% of all pregnancies. Hallmarked by hypertension and endothelial dysfunction the origin of the disease remains obscure, though it is generally accepted that placental insufficiency/ischemia is a central cause. In response, the placenta secretes pathogenic factors, in particular the anti-angiogenic protein sFlt-1. Currently, there is no effective therapy for the management of the preeclampsia patient. We have recently produced a novel synthetic peptide based on placental growth factor (PlGF) which is maternally restricted by fusion to the synthetic carrier elastin like polypeptide (ELP). Here, we describe its in vivo pharmacokinetics and biodistribution.

Methods:
Fluorescently labeled ELP-PLGF was administered i.v. and blood sampled serially to determine clearance kinetics. Long-term pharmacokinetics and biodistribution was performed after subcutaneous administration of labeled peptide. Measurements were made on serially drawn blood, and in the whole animal by in vivo imaging.

Results:
ELP-PlGF exhibited markedly more favorable pharmacokinetics than the normal half life of PlGF, with a terminal half-life of ~10 hours as opposed to ~30 minutes for PlGF alone. Chronic administration found highest levels accumulating in placenta and kidney (two favorable targets for preeclampsia) and liver. A single subcutaneous administration at 100mg/kg resulted in sustained therapeutic plasma concentrations for over 10 days.

Conclusion:
These data demonstrate that ELP-PlGF has favorable pharmacokinetic and biodistribution profiles. Previous data suggest ELP-PlGF directly antagonizes sFlt-1 in culture. Future studies to assess the in vivo effectiveness of ELP-PlGF in managing placental ischemia induced hypertension and endothelial dysfunction are currently in progress.

Acknowledgment:
This work was supported by NIH grants R0121527 (GLB), T32HL105324 (OCL), P01HL51971, P20GM104357 (EMG), and R00HL116774 (EMG)
Abstract 037: Role of Pro-opiomelanocortin (POMC) Specific PTP1B in Differential Regulation of Blood Pressure, Liver Lipid Accumulation and Glucose Tolerance in Response to a High Fat Diet
Authors: Aberdein Nicola , do Carmo Jussara M , Wang Zhen , Fang Taolin , de Lara Cecilia P , Hall John E
Published: June 2021

Obese subjects are often resistant to leptin’s metabolic effects although blood pressure (BP) and sympathetic nervous system responses appear to be preserved. Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of leptin signaling, may play a role in promoting this selective leptin resistance and causing metabolic dysfunction in obesity. Our previous studies suggest that the chronic BP responses to leptin are mediated via activation of pro-opiomelanocortin (POMC) neurons. The goal of this study was to determine if PTP1B in POMC neurons differentially controls metabolic functions and BP in mice fed a high fat diet (HFD). Male mice with POMC specific PTP1B deletion (POMC/PTP1B
-/-
) and littermate controls (PTP1B
flox/flox
) were fed a HFD from 6 to 22 wks of age. Baseline BP after 16 weeks of a HFD (95±2 vs. 95±3 mmHg) and BP responses to acute stress (Δ32±0 vs. Δ32±6 mmHg), measured by telemetry, were not different in POMC/PTP1B
-/-
compared to control mice, respectively. Heart rate (HR) was not different in POMC/PTP1B
-/-
and control mice during acute stress (699±4 vs. 697±15 bpm, respectively). Total body weight (TBW) and fat mass were reduced at 20 weeks of age in POMC/PTP1B
-/-
compared to controls (36.7±0.1 vs. 42.0±1 g TBW and 12.7±0.4 vs. 16.1±1.0 g fat mass, respectively). Liver weight of POMC/PTP1B
-/-
mice was less than in controls, and this was evident even when liver weight was normalized as % of TBW (4.5±0.2 vs. 5.0±0.2 %). POMC/PTP1B
-/-
males had reduced liver lipid accumulation compared to controls as measured by EchoMRI (0.08±0.03 vs. 0.15±0.03 g/g liver weight). Glucose tolerance was also improved by 46% in POMC/PTP1B
-/-
compared to controls as measured by AUC, 25856±1683 vs. 47267±5616 mg/dLx120min, respectively. These findings indicate that PTP1B signaling in POMC neurons plays a crucial role in regulating liver lipid accumulation and glucose tolerance but does not appear to mediate changes in BP or BP responses to acute stress in mice fed a high HFD (supported by NHLBI-PO1HL51971 and NIGMS P20GM104357)
Abstract 038: Hyperglycemia Mediated Cardiac Damage in High-Fat Fed E487K Aldehyde Dehydrogenase 2 (ALDH 2*2) Mutant Diabetic Mice: Role for ALDH2 Activation
Authors: Pan Guodong , Palaniyandi Suresh
Published: June 2021

Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme in the heart, detoxifies reactive aldehydes and protects heart from oxidative stress. East Asians (~700 million) are carriers of E487K point mutation of ALDH2 (ALDH2*2) with intrinsically low ALDH2 activity. ALDH2*2 is associated with increased maternal inheritance of diabetes mellitus (DM), and DM-induced neuropathy and vasculopathy. However the pathophysiology of diabetic cardiac damage.is not studied in ALDH2*2 carriers. DM is a polygenic disease and DM-induced cardiac damage may be multifactorial. However we hypothesis that hyperglycemia-induced oxidative stress mediated 4-hydroxy-2-nonel (4HNE) toxicity contributes to the cardiac damage in ALDH2*2 mutant mice (low intrinsic ALDH2 activity) with type-2 diabetes. We induced type-2 diabetes by feeding high-fat diet and found they developed hyperglycemia (blood glucose (BG) levels increased to 357 ± 100 mg/dl
vs
137 ± 7 mg/dl) and insulin resistance as measured by glucose tolerance test (GTT) (BG levels 408 ± 50 mg/dl
vs
165 ± 18 mg/dl at 2 hours after GTT). To delineate the role of hyperglycemia, we treated the diabetic mice with a sodium-glucose co-transporter 2 (SGLT2) inhibitor, Empaglifuzin (EMP) (3mg/kg/day) or Vehicle for 8 weeks. EMP reduced BG levels from 502 ± 75 mg/dl to 193 ± 50 mg/dl by enhancing urinary glucose excretion. Surprisingly EMP reversed insulin resistance as maintained similar BG levels before and after 2 hours of GTT; 190 ± 23 mg/dl vs 188 ± 16 mg/dl. EMP also increased ALDH2 activity to 22 ± 8 % from 7 ± 3 % and 4HNE protein adduct levels. Finally EMP improved cardiac function i.e. % fractional shortening (FS) is increased to 70 ± 4 compared to 53 ± 10. Our data suggested hyperglycemia partially contribute to the diabetic cardiac damage via increasing 4HNE protein adducts. Alda-1 (10 mg/kg/day) treatment further augmented ALDH2 activity, reduced 4HNE adducts and improved cardiac function in EMP-treated ALDH2*2 mice. Thus hyperglycemia mediated secondary events in type-2 DM are significant pathomechanism of the cardiac damage. In conclusion, we propose ALDH2 activation may ameliorate diabetic patients from cardiac complications who receive glucose lowering treatments.
Abstract 042: Selective Maternal Hepatic Silencing of Angiotensinogen in Reduced Uterine Placental Perfusion Pressure Rats Improves Blood Pressure and Fetal Weights
Authors: Cunningham Mark W , Haase Nadine , Faulkner Jessica L , Ibrahim Tarek , Vaka Venkata Ramana , Dechend Ralf , Foster Don , LaMarca Babbette
Published: June 2021

Women with Preeclampsia (PE), a form of new onset hypertension during pregnancy, exhibit alterations in the renin angiotension system (RAS). These differences include angiotensin II type 1 receptor agonistic autoantibodies (AT1-AA), ANG II sensitivity, and increased oxidation and levels of angiotensinogen (AGT). While antihypertensive drugs are used in pregnancy, their use is associated with reduced fetal growth, and RAS blockade is specifically contraindicated due to fetotoxicity. AGT is the precursor to ANG II and the initial substrate of the RAS pathway. Genetic mutations that result in an over-expression of AGT are strongly associated with hypertension. We have previously shown that siRNA targeting maternal hepatic AGT in a transgenic model of preeclampsia (hAGT x hREN) resulted in lower blood pressure, a 90% reduction in AT1-AA activity, and normalized IUGR. The purpose of the present study is to examine the effect of silencing maternal hepatic AGT on maternal blood pressure and fetal growth in the reduced uterine placental perfusion pressure (RUPP) model of PE.

Methods:
Pregnant Sprague Dawley rats were divided into 4 groups: normal pregnant (NP, n=7); RUPP (n= 6); RUPP + siRNA (n =7); NP + siRNA (n=7). On day 12 of gestation (GD), siRNA was subcutaneously injected into rats (10mg/kg), GD14 the RUPP surgery was performed, GD18 carotid catheters inserted, and GD19 conscious blood pressure (MAP) and fetal weight was recorded.

Results:
MAP was elevated in RUPP vs. NP (134±6 vs. 101±4 mmHg, p< 0.05), but was lower in RUPP + siRNA (117±4 mmHg) and unchanged in the NP+siRNA (92±3 mmHg) group compared to NP. Average fetal weights by group were 2.30±0.09 g (NP), 1.89±0.10 g (RUPP), 2.38±0.09 g (NP+siRNA), and 2.12±0.06 (RUPP+siRNA). A one-way ANOVA indicates a significant reduction in pup weights between NP and RUPP, but not NP+siRNA and RUPP+siRNA, indicating administration of siRNA to RUPP rats prevented the decrease in fetal weight.

Conclusion:
Administration of AGT siRNA to RUPP rats blunts the increase in blood pressure and prevents a decrease in fetal weights in response to placental ischemia and thus could be a potential therapy for management of preeclampsia and other hypertensive disorders of pregnancy. Research Supported by Alnylam & T32HL105324,
Abstract 047: Activation of Sirt1 Deacetylase Attenuates Klotho Deficiency-induced Arterial Stiffness and Hypertension
Authors: Gao Amy , Ali Quaisar , Zuo Zhong , Lin Yi , Lei Han , Sun Zhongjie
Published: June 2021

Objective:
Arterial stiffness is an independent risk factor for stroke and myocardial infarction. This study was designed to investigate the role of SIRT1, an important deacetylase, and its relationship with Klotho, a kidney-derived aging-suppressor protein, in the pathogenesis of arterial stiffness and hypertension.

Methods and Results:
We found that the serum level of Klotho was decreased by nearly 45% in patients with arterial stiffness and hypertension. Interestingly, Klotho haplodeficiency caused arterial stiffening and hypertension, as evidenced by significant increases in pulse wave velocity (PWV) and blood pressure (BP) in Klotho-haplodeficient (

KL
+/-

) mice. Notably, the expression and activity of SIRT1 were decreased significantly in aortic endothelial and smooth muscle cells in
KL
+/-
mice, suggesting that Klotho deficiency downregulates SIRT1. Treatment with SRT1720 (15 mg/kg/day, IP), a specific SIRT1 activator, abolished Klotho deficiency-induced arterial stiffness and hypertension in

KL
+/-

mice. Klotho deficiency was associated with significant decreases in activities of AMP-activated protein kinase alpha (AMPKα) and endothelial nitric oxide synthase (eNOS) in aortas, which were abolished by SRT1720. Furthermore, Klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and enhanced elastin fragmentation in the media of aortas. These Klotho deficiency-associated changes were blocked by SRT1720.

Conclusion:
This study provides the first evidence that Klotho deficiency downregulates SIRT1 activity in arterial endothelial and smooth muscle cells. Pharmacological activation of SIRT1 is likely to be an effective therapeutic approach for arterial stiffness and hypertension.
Abstract 049: Phosphodiesterase-3a Catalytic-domain Mutation and Hypertension
Authors: Bähring Sylvia , Schächterle Carolin , Aydin Atakan , Klussmann Enno , Luft Friedrich C
Published: June 2021

We recently discovered phosphodiesterase-3A (PDE3A) mutations causing a 50 mm Hg increase in blood pressure and stroke >50 years, as the first non-salt form of Mendelian genetic hypertension, autosomal-dominant hypertension with brachydactyly (HTNB). The mutations cause increased PDE3A phosphorylation and higher cAMP affinity. We now have found a completely different PDE3A mutation causing a similar syndrome in a New Zealand pedigree. The mutation resides in the enzyme’s catalytic domain, results in an arginine-to-cysteine substitution, and represents a more direct mechanism of PDE3A activation. For Michaelis-Menten kinetics of cAMP hydrolysis, we transfected HEK293 cells transiently expressing Flag-tagged versions of PDE3A1, PDE3A2, or PDE3A3 mutant vs. wildtype and stimulated with forskolin and phorbol-12-myristate-13-acetate (PMA) to enhance intrinsic phosphorylation. Vmax and Km (Michaelis constant) were calculated using GraphPad Prism software to reveal the maximum cAMP turnover rate at saturated substrate concentration and the affinity of cAMP to wildtype and mutated PDE3A1, PDE3A2 and PDE3A3. For PDE3A1 hydrolytic activity (triplicate), we observed: Vmax Km Wildtype 7.5 340 Wildtype+forskolin/PMA 7.2 203 Mutant 6.6 116 Mutant+forskolin/PMA 6.3 81 The dramatically lower Km of mutant PDE3A indicates a substantially greater affinity for cAMP consistent with gain-of-function. These data underscore the importance of PDE3A to high blood pressure by means of a different, novel genetic mechanism directly implicating the catalytic domain.
Abstract 056: Isolevuglandin-modified Phosphatidylethanolamines Generate Hypertension and Vascular Dysfunction in Mice
Authors: Pandey Arvind K , Itani Hana A , Xiao Liang , Kirabo Annet , Dikalov Sergey , Davies Sean , Harrison David G
Published: June 2021

Oxidative stress is an important contributor to hypertension. Our group has shown that oxidative stress generates isolevuglandins (isoLG) that modify proteins and that these isoLG-modified proteins seem to act as neoantigens to promote hypertension. In addition to proteins, isoLG can also modify phosphatidylethanolamines (PEs). IsoLG-modified PE (IsoLG-PE) can in turn activate immune cells via reactions with toll-like receptors and downstream pathways including NFκB. The role of isoLG-PE in hypertension has not yet been defined. We have previously shown that the enzyme N-acyl-phosphatidylethanolamine-phospholipase D (NAPE-PLD) cleaves isoLG-PE, and mice lacking this enzyme have elevated cellular levels of isoLG-PE. To assess whether loss of NAPE-PLD and resulting increase in isoLG-PE contributes to hypertension, we infused wild type (WT) and NAPE-PLD
-/-
mice with low dose Ang II. We found baseline blood pressure was elevated in NAPE-PLD
-/-
mice compared to WT mice (128 ± 3 vs 111 ± 2 mmHg, p = 0.005) and that the increase in BP to low dose Ang II infusion (140 ng/kg/min) was augmented in NAPE-PLD
-/-
compared to WT mice (146 ± 9 vs 127 ± 4 mmHg, p=0.045). Endothelium-dependent vascular relaxation of the mesenteric arterioles to acetylcholine was impaired in NAPE-PLD
-/-
compared to WT mice (maximum relaxation of 43% ± 6% vs 53% ± 5%, p= 0.004), but endothelium-independent responses to sodium nitroprusside were similar in both groups (82% ± 2% in NAPE-PLD
-/-
mice vs 81% ± 4% in WT mice). Aortic adventitial collagen content by planimetry was likewise increased in NAPE-PLD
-/-
versus WT mice following low-dose Ang II. These studies in mice lacking NAPE-PLD suggest that in addition to exerting its effects via protein modification to form neo-antigens, the reaction of isoLG with PE and related phospholipids can augment hypertension, alter endothelium-dependent vasodilatation and promote vascular fibrosis.
Abstract 067: Unexpected Actions of the Endothelial-EP
4
Receptor for PGE
2
to Promote Hypertension
Authors: Yang Ting , Herrera Marcela , Sparks Matthew A , Manning Michael , Koller Beverly H , Coffman Thomas M
Published: June 2021

Prostaglandin E2 (PGE
2
) is a major prostanoid produced by the kidney with vasodilator and natriuretic actions and its actions are mediated by four distinct E-prostanoid (EP) receptor isoforms: EP
1
-EP
4
. The EP
4
receptor (EP
4
R) has multiple actions that could impact blood pressure (BP) by triggering macula densa stimulation of renin, inducing vasodilation, and inhibiting epithelial sodium transport. Accordingly, we examined the role of EP
4
R on BP regulation by generating EP
4
R-deficient mice. Because deletion of EP
4
R in utero causes peri-natal mortality due to persistent patent ductus arteriosus, we carried out conditional deletion using an

EP
4
flox/flox

mouse line. We first generated mice completely lacking EP
4
R in all tissues (TBKO) using a tamoxifen-inducible transgene driving
Cre
expression in all tissues. Resting mean arterial pressure (MAP) measured by radiotelemetry tended to be elevated in TBKOs compared to controls (106±2 vs 111±2 mmHg; p=0.06). In addition TBKOs showed exaggerated salt sensitivity and enhanced hypertensive response to chronic Ang II infusion compared to controls (MAP increase: 25±3 vs. 37±2 mmHg; p<0.05). To determine whether altered BP responses in the TBKOs were due to elimination of EP
4
R-depedent actions in vascular smooth muscle cells (VSMCs) or in endothelial cells (ECs), we generated mice lacking EP
4
R in VSMCs (SMKO) or ECs (ECKO) using

EP
4
flox/flox

and transgenic mice with tamoxifen-inducible expression of
Cre
limited to VSMCs or ECs. Resting MAP in SMKO mice was significantly reduced compared to controls (109±1 vs. 104±2 mmHg; p<0.05), but salt sensitivity and Ang II-dependent hypertension were unaffected. Although no statistically significant differences in baseline MAP or salt sensitivity were observed between ECKOs and controls, the hypertensive response to AngII infusion was significantly reduced in ECKOs (MAP increase: 31±3 vs 24±2 mmHg; p<0.05). In summary, our work suggests a complex role for PGE
2
acting via its EP
4
R in BP regulation, with a major effect to promote resistance to hypertension, apparent in the TBKOs. However, we have also uncovered an unexpected and opposing effect of EP
4
R in endothelium to promote hypertension.
Abstract 069: β-Catenin Signaling Contributes to Angiotensin II-Induced Hypertension via Activation of Sodium Transporters in the Distal Nephron
Authors: Lu Xiaohan , Peng Kexin , Wang Fei , Yang Tianxin
Published: June 2021

β-Catenin signaling plays an important role in regulation of development as well as tissue homeostasis. Recently, we have shown that this pathway is involved in regulation of collecting duct (CD) function. Here, we examined the role of β-catenin signaling in AngII-induced hypertension. C57/BL6 mice were administered for 7 d with vehicle, AngII alone (300 ng/kg/min) or in combination with an inhibitor of β-catenin pathway, ICG-001 (50 mg/kg/d) both via an osmotic mini-pump. Radiotelemetry demonstrated that ICG-001 effectively attenuated AngII-induced hypertension (MAP on day 7: 112.5 ± 2.02 in Control group vs. 137±2.07 in AngII group vs. 119.2±2.26 mmHg in AngII/ICG-001 group, n = 7-9, p<0.05) accompanied with a 34% (34 of 100) increase in 24-h urine volume and a 35% (35 of 100) increase in 24-h urinary Na+ excretion. Following AngII treatment, urinary renin activity and renin content both exhibited a more than 10-fold increase which was completely blocked by ICG-001. AngII infusion selectively elevated α-ENaC protein abundance in the renal medulla but not in the renal cortex; the renal medullary upregulation of α-ENaC protein expression was attenuated by ICG-001. Similarly, renal cortical NCC and NKCC2 protein expression was elevated by AngII infusion; this upregulation of NCC and NKCC2 protein expression was again abolished by ICG-001. In contrast, NHE3 protein abundance remained constant. In cultured mpkCCD cells transfected with a β-catenin-driven luciferase construct, AngII treatment at 500 nM for 24 h induced a 10-fold increase in the reporter activity which was sensitive to ICG-001 (1 μM). In these cells, the AngII treatment induced amiloride-sensitive Na
+
transport as assessed by epithelial volt-ohmmeter, which was completely blocked by ICG-001. Protein expression of α-ENaC exhibited the similar pattern of changes as ENaC activity whereas the expression of β- or γ-ENaC remained constant. ICG-001 was not associated with noticeable toxicity in vivo or in vitro. In summary, these results suggest that β-catenin signaling mediates AngII-induced hypertension at least in part through regulation of intrarenal renin response and the expression of Na+ transporters in the distal nephron.
Abstract 084: Sodium Butyrate and Retinoic Acid Attenuate Renal Inflammation and Fibrosis in
Npr1
Gene-targeted Haplotype Mice: Role of NF-κB Signaling
Authors: Kumar Prerna , Periyasamy Ramu , Gogulamudi Venkateswara , Pandey Kailash N
Published: June 2021

The objective of the present study was to determine the effect of a hybrid drug of sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor and all-trans retinoic acid (ATRA) on the attenuation of renal inflammation and fibrosis in
Npr1
gene-disrupted mutant mice. Adult (18-20 week old) male
Npr1
gene-disrupted heterozygous (1-copy;
Npr1
+/-
) wild-type (2-copy;
Npr1
+/+
), and gene-duplicated (3-copy;
Npr1
++/+
) mice were treated with ATRA-NaBu hybrid drug (1.0 mg/kg/day) by intraperitoneal injections for 2-weeks. A significant decrease in systolic blood pressure was observed in ATRA-NaBu-treated haplotype
Npr1
+/-
mice compared with untreated controls (treated, 113.3 ± 1.5 vs. control, 130.4 ± 1.9; p < 0.01). After treatment with ATRA-NaBu, a marked reduction in tubulo-interstitial fibrosis (50%, p < 0.001) and decreased renal collagen type I alpha 2 expression by 55% (treated, 25.9 ± 1.2 vs. control, 57.2 ± 2.9, p < 0.001) was observed in
Npr1
+/-
mice. Treatment with ATRA-NaBu also increased creatinine clearance (ml/24 h) in
Npr1
+/-
mice (245.7 ± 24.3 vs. control, 83.8 ± 3.9). Similarly, higher urinary albumin to creatinine ratio was detected in
Npr1
+/-
mice (0.84 ± 0.03) vs. control (0.35 ± 0.03; p < 0.01) and a complete reversal was observed in drug-treated
Npr1
+/-
mice (0.39 ± 0.05). Significant decreases were observed in renal (pg/mg protein) tumor necrosis factor-alpha (TNF-α) (4.1 ± 0.7 vs. control, 27.2 ± 2.6; p < 0.01) and interleukin (IL)-6 (11.8 ± 0.8 vs. control, 59.9 ± 3.6; p < 0.01) in ATRA-NaBu-treated
Npr1
+/-
mice. Western blot analyses showed significant reduction in renal TNF-α and IL-6 protein expression by 54%, (p < 0.001) and 61%, (p < 0.01), respectively, in ATRA-NaBu-treated
Npr1
+/-
mice. There was 49% increase in renal NF-κB (p65) DNA binding activity in
Npr1
+/-
mice and 51% lower activity in
Npr1
+/++
mice compared with wild-type mice. Western blot analysis revealed distinctly higher levels of renal NF-κB (p65) protein expression in
Npr1
+/-
mice and reduced levels in
Npr1
+/++
mice compared with wild-type controls. The present results provide direct evidence that ATRA-NaBu hybrid drug acts as a potent anti-inflammatory agent, which will have important implications in the pathophysiology of renal injury and hypertension.
Abstract 097: Effects of Lysine-specific Demethylase 1 Deficiency on Aldosterone Production and Salt-sensitive Hypertension in Female Mice
Authors: Huang Yuefei , Yao Tham M , Loutraris Paul , Rangel Isis K , Ting Pei Yee , Tan Jia Wei , Garza Amanda E , Romero Jose R , Pojoga Luminita H , Adler Gail K , Williams Gordon H
Published: June 2021

Lysine-Specific Demethylase1 (LSD1) is an epigenetic factor modulated by salt intake. Previously, we documented the male heterozygote LSD1 knockout mice (LSD1+/-) had dysregulation of aldosterone (ALDO) production on a liberal salt diet (1.6% Na+) associated with salt-sensitive hypertension. This study assessed if: 1) female LSD1+/- mice have a similar phenotype; and 2) the effect of aging on this phenotype.

Methods:
Female LSD1+/- and wild type mice (LSD1+/+) were randomly assigned for sacrifice at the ages of 18-week, 52-week, and 75-week and the following were assessed at each time point: blood pressure (BP); plasma renin activity (PRA) and ALDO; urine albumin; and
ex vivo
ALDO production from isolated adrenal zona glomerulosa cells.

Results:
BP and urine albumin in the LSD1+/- compared to the LSD1+/+ were not different at any age (Table). However, the LSD1+/- had greater ALDO/PRA ratios at 18 weeks compared with the LSD1+/+, but lower ALDO levels and
ex vivo
ALDO production at 52 and 75 weeks. Associated with this phenotype, the LSD1+/- showed significantly higher rate of all-cause mortality than the LSD1+/+.

Conclusion:
Lack of LSD1 caused dysregulation of ALDO production in both male and female mice. But the cardiovascular outcomes are different. The LSD1+/- females in contrast to males do not develop hypertension or albuminuria even at 75 weeks of age. However, the females do die at a faster rate than the males of a variety of causes. Thus, there is considerable sexual dimorphism in the pathogenesis of cardiovascular outcomes associated with dysregulation of adrenal ALDO production mediated by lack of LSD1.
Abstract 098: A Novel Mechanism for Buffering Dietary Salt in Humans: Effects of Salt Loading on Skin Sodium, VEGF-C and Blood Pressure
Authors: Selvarajah Viknesh , Maki-Petaja Kaisa , Pedro Liliana , Bruggraber Sylvaine F , Brown Morris J , McEniery Carmel M , Wilkinson Ian B
Published: June 2021

Introduction:
Dietary sodium is an important trigger for hypertension. Animal studies show that the skin buffers dietary salt and salt-loading induces lymphangiogenesis mediated by VEGF-C, maintaining normal BP. Our primary objective was to determine whether increased skin Na
+
on salt loading attenuates the expected rise in blood pressure.

Methods:
We assessed skin and urine electrolytes, systemic haemodynamics, ambulatory BP and plasma VEGF-C in 48 healthy participants. Participants were placed on a low salt diet (70mmol sodium/day) and received placebo and slow sodium (200mmol daily for 7 days) in a randomised order. Skin Na
+
and K
+
concentrations were measured in mg/g tissue by inductively coupled plasma optical emission spectroscopy. Results were expressed as the ratio of Na
+
:K
+
to correct for variability in sample hydration. Plasma VEGF-C was analysed by ELISA.

Results:
Mean age was 30 ± 8 years (24 male). 24-hr urine Na
+
was higher following salt than placebo in males (221.9 ± 16.5 vs. 86.3 ± 10.3mmol, p < 0.001) and females (227.1 ± 19.9 vs. 60.0 ± 6.8mmol, p < 0.001). In males, skin Na
+
:K
+
was higher after salt loading (2.87 ± 0.12 vs. 2.59 ± 0.09 mg/g , p = 0.008), but not significantly different in females (3.36 ± 0.12 vs. 3.23 ± 0.10 mg/g, p = 0.49). Females showed a significant increase in 24-hr MAP with salt loading (88 ± 1 vs. 85 ± 1 mmHg, p=0.004) but not males (90 ± 2 vs. 90 ± 2 mm Hg, p = 0.91). In males only, skin Na
+
:K
+
correlated with supine MAP post-placebo (r = 0.52, p = 0.004) and post-salt (r = 0.51, p = 0.01). No significant change was noted in plasma VEGF-C.

Conclusions:
Salt loading causes an increase in BP in healthy females but not in males, whilst skin sodium rises in males. This indicates that the skin may buffer the haemodynamic consequences of increased salt intake in a gender-specific manner.
Abstract 109: Association of Endothelial Dysfunction with Non-invasive Measures of Vascular Stiffness in Young-middle Aged Non-hypertensive Individuals
Authors: Cheng Cynthia , Townsend Raymond , Chirinos Julio A , Keith Scott
Published: June 2021

Arterial stiffness and enhanced wave reflections independently predict cardiovascular risk. Wave reflections augment central (aortic) pulse pressure (PP), an index of arterial stiffness, and systolic pressure. Increased wave reflections and PP have previously been associated with endothelial dysfunction in hypertensive and healthy middle-aged adults. The study objective was to determine whether endothelial dysfunction is associated with PP and other measures of vascular stiffness in young normotensive and prehypertensive subjects.

We measured office, central, and 24-hour measurements in 102 (64 female, 38 male) non-hypertensive, non-diabetic participants. Endothelial function was assessed non-invasively using post-ischemic reactive hyperemia with strain-gauge plethysmography.

The racially diverse subject pool was comprised of 60% Caucasians, 18% African Americans, and 24% Asians, with mean age 30, mean BMI 25.6, mean office SBP/DBP = 110 ± 13 mm Hg/70 ± 9 mm Hg. Endothelial function was highly associated with office (β= - 4.2 mm Hg, p<0.001) and 24-hour PP (β= - 1.4 mm Hg, p=0.008), along with central measures of wave reflection: ((augmentation pressure (β= - 2.1 mm Hg), augmentation index (β= - 3.7): both p<0.001). Beta values correspond to the change noted for one standard deviation in endothelial function.

In conclusion, endothelial dysfunction is significantly and consistently associated with arterial stiffness and increased wave reflections in young non-hypertensive adults. Identification of endothelial dysfunction in otherwise healthy young individuals may provide an opportunity to reduce vascular stiffness and associated cardiovascular risk.
Abstract 110: Vascular Smooth Muscle Sirtuin-1 Protects Against Diet-Induced Aortic Stiffness
Authors: Fry Jessica , Al Sayah Leona , Weisbrod Robert , Van Roy Isabelle , Weng Xiang , Cohen Richard , Bachschmid Markus , Seta Francesca
Published: June 2021

Arterial stiffness (AS), a major cardiovascular risk factor, develops within two months in mice fed a high fat, high sucrose diet (HFHS), serving as a model of human metabolic syndrome, and is associated with functional impairment of vascular smooth muscle (VSM) cells. Sirtuin1 (SirT1) is a NAD
+
-dependent deacetylase induced in response to cellular stresses. Our goal was to study the effects of VSM SirT1 on AS in the context of diet-induced metabolic syndrome.

VSM specific genetic SirT1 over-expression (SMTG) prevented AS induced by HFHS, measured
in vivo
by pulse wave velocity (PWV) over 8 months. Resveratrol or S17834, two polyphenolic compounds known to activate SirT1, prevented HFHS-induced AS and were mimicked by global SirT1 over-expression (SirBACO), without evident metabolic improvements (HFHS-induced weight gains or response to a glucose tolerance test remained unchanged). Additionally, HFHS-induced PWV increases were reversed by one-week treatment with a specific, small molecule SirT1 activator (SRT1720). Overnight fasting acutely decreased PWV in mice fed HFHS for 8 months, but not in mice lacking SirT1 in VSM (SMKO). These beneficial effects of pharmacological, genetic or fasting-induced SirT1 activation against AS, were associated with a decrease in NFκB activation and VCAM-1 and p47phox protein expressions, in aorta and VSM cells.

In conclusion, VSM SirT1 activation decreases AS in the setting of obesity by stimulating anti-inflammatory and anti-oxidant pathways in the aortic wall. SirT1 activators may represent a novel therapeutic approach to prevent AS and associated CV complications in overweight/obese individuals with metabolic syndrome.
Abstract 113: Ambulatory Blood Pressure in Hypertensive Patients with Inclusion Criteria for the SPRINT Trial
Authors: De La Sierra Alejandro , Banegas Jose R , Divison Juan A , Gorostidi Manuel , Vinyoles Ernest , De La Cruz Juan J , Segura Julian , Ruilope Luis M
Published: June 2021

The SPRINT trial has demonstrated the benefit of intensive BP reduction in hypertensive patients at high cardiovascular risk. Values of ABPM are of potential interest in such patients to better select those who will benefit of a lower BP target. We aimed to evaluate ABPM values in a large cohort of patients potentially candidates (meeting inclusion criteria) for the SPRINT trial. Moreover, in patients on antihypertensive therapy who also fulfill SPRINT criteria, except for clinic SBP ≥ 130 mmHg, we evaluated 24-hour SBP values among those who had clinic SBP < 120; between 120 and 139, or ≥ 140. From the database of the Spanish ABPM Registry containing 115708 patients, we identified 39132 (34%, 51% women, mean age 65 years) who fulfill both inclusion and exclusion criteria of the SPRINT trial. Mean values of clinic SBP were 151±11 mmHg, whereas corresponding values for 24-h SBP were 130±13 mmHg. Overall, 52% of patients had 24-h SBP below 130 mmHg. The proportion varied from 69% in those with clinic BP 130-139 to 34% in those with clinic BP ≥ 170 mmHg. Among 34328 treated patients who fulfilled SPRINT inclusion criteria (except for clinic BP ≥ 130), 1014 (3%) had clinic SBP < 120 mmHg, and 5330 (16%) values between 120 and 139 mmHg. The remaining 27984 patients were not controlled and had clinic SBP ≥ 140 mmHg. Values of 24 h SBP below 130 mmHg were seen in 88% of those with clinic BP < 120, in 74% of those with clinic BP 120-139, and in 47% of those with clinic BP ≥ 140 mmHg. The corresponding proportion of patients having 24-h SBP < 100 mmHg were 7.7%, 1.1%, and 0.6%. We conclude that ABPM assessment could be necessary in the evaluation of hypertensive patients at high CV risk before targeting the BP goal, as roughly half of them may have normal values of 24-h SBP (< 130 mmHg). In addition, targeting clinic BP below 120 mmHg is accompanied by 8% of patients with 24-h SBP below 100 mmHg.
Abstract 125: Interleukin 6 Activates the Mineralocorticoid Receptor via Rac1, Increasing Sodium Chloride Cotransporter Expression and Activity
Authors: Wynne Brandi M , van Elst Henrieke J , Kronk Trinity A , Hecht Gillian , Al-Khalili Otor , Ko Benjamin , Eaton Douglas C , Hoover Robert S
Published: June 2021

Hypertension is characterized by increased sodium (Na
+
) reabsorption along the aldosterone-sensitive distal nephron (ASDN), as well as a chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na
+
reabsorption within the ASDN does not always correlate with increases in serum aldosterone (Aldo), the hormone that modulates Na
+
reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased MR-mediated Na
+
reabsorption may occur independent of Aldo stimulation is critical. We hypothesize that IL-6 transactivates the MR via Rac1, increasing sodium chloride cotransporter (NCC) expression and activity. To demonstrate IL-6-mediated MR activation via Rac1, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs, treated with vehicle, Aldo (100nM), IL-6 (100ng/mL) and/or Rac1 inhibitor (EHT1864, 20uMol), and luciferase assays performed (48hrs). To determine if IL-6 increases NCC expression,
in vivo
, we perfused kidneys with IL-6 (intra-renal, 16ng/hr, 1-3d) and the cortex was isolated for WB analysis of total NCC expression. To determine direct IL-6 mediated effects on NCC activity, we performed thiazide-sensitive
22
Na
+
-uptake studies in cell monolayers in presence of: vehicle, IL-6 (100ng/mL for 45min or 6hrs) or IL-6+MR antagonist (spironolactone, 20uMol). IL-6 treatment significantly increased luciferase activity (2.6±0.9 fold/MRE-only), which was reduced with Rac1 inhibition (0.2±0.08, p<0.01, n=8-10) demonstrating a Rac1-dependent activation of MR via IL-6. In the kidney cortex, IL-6 infusion increased total NCC expression (>2.5 fold) after day 3, as compared to vehicle. In addition,
22
Na
+
-uptake studies (n=6) revealed an IL-6-mediated increase in Na
+
transport (1927±40 nmol/mg/20min
vs.
1503±7 nmol/mg/20min, p<0.0001), which was reduced with the MR antagonist (1721±25nmol/mg, p<0.001). These data suggest that IL-6 activates the MR via Rac1, leading to increased NCC expression and activity. These data provide evidence for alternate mechanisms of increased ASDN Na
+
uptake during inflammatory and hypertensive conditions, independently of Aldo-mediated signaling.