bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P039: Testosterone Increases BP in Male SHR by Activating the Renin-angiotensin System: A Cautionary Tale for “Low T” Supplements
Authors: Maranon Rodrigo O , Yanes Cardozo Licy L , Dalmasso Carolina , Patil Chetan N , Zhang Huiming , Harris Andrew , Reckelhoff Jane F
Published: July 2021

Testosterone supplements are widely prescribed for men in the US to improve overall quality of life by increasing libido and sexual performance and protecting against osteoporosis. Whether testosterone supplements affect CVD either positively or negatively is not clear since study results are controversial. Previously, we showed that chronic testosterone supplements cause hypertension in obese male Zucker rats but improves inflammation and metabolic syndrome symptoms. We also found that testosterone supplements in young male SHR further increase their BP, and that castration attenuates the hypertension. In the present study we followed up on these previous studies and tested the hypothesis that, because the SHR are lean, chronic testosterone supplements will increase lean body mass but increase the BP via a renin angiotensin system (RAS) mechanism. Male SHR were treated with testosterone (8 mg/10 mm silastic pellet implanted sc) or placebo (empty pellets sc), beginning at 12 weeks of age for 8 weeks. Fat mass and lean mass were measured by ECHO MRI. Contrary to our hypothesis, testosterone supplements had no effect on lean mass, but reduced fat mass by 49% (4.9±0.6 vs 2.5±0.4 %BW, p<0.05). Baseline mean arterial pressure (MAP by telemetry) was higher in testosterone supplemented rats than placebo controls (157±0.4 vs. 147±0.7 mmHg, p<0.05). Enalapril (200 mg/L) reduced MAP by 25% in testosterone group (157±0.4 vs 118±6 mmHg, p<0.05) and by 14% placebo rats (148±0.5 vs 127±5 mmHg, p<0.05). MAPs after enalapril were similar between placebo and testosterone-treated rats. These data suggest that the RAS contributes to the elevated BP in testosterone supplemented male SHR. The data also suggest that caution should be taken in prescribing hypertensive men with testosterone supplements. Supported by NIH-R01HL66072, PO1HL51971 (JFR), 14POST18640015 (ROM), EFF Endocrine Res Grant (LLY).
Abstract P040: Role of 20-HETE in Elevated Blood Pressure in Hyperandrogenemic Female Rats, a Model of Polycystic Ovarian Syndrome
Authors: Patil Chetan N , Dalmasso Carolina , Maranon Rodrigo O , Zhang Huimin , Roman Richard J , Reckelhoff Jane F
Published: July 2021

Polycystic ovary syndrome (PCOS) is the most common reproductive disorder in premenopausal women, is characterized by hyperandrogenemia, metabolic syndrome and inflammation. They also exhibit elevated blood pressure (BP) but may not be treated since they do not meet the criteria for hypertension (BP>130/90 mm Hg). We have characterized a female rat model of hyperandrogenemia (HAF) using dihydrotestosterone (DHT) that mimics many characteristics of women with PCOS. In the present study we tested the hypothesis that androgen-induced upregulation of the cytochrome P450 4A2 isoform (CYP4A2) and the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) in renal microvasculature contributes to the elevated BP in HAF rats. Female rats of SS.5BN consomic strain (wild type) rats and CYP4A2-/- rats on this same background were implanted with DHT (7.5mg/90d) or placebo pellets (n=5-8/grp) beginning at 6 wks of age; pellets were changed every 85 d. At 14 wks of age, rats were implanted with radiotelemetry transmitters, and mean arterial pressure (MAP) was measured for 10 days. Endogenous 20-HETE levels were measured using LC-MS in renal microvessels isolated using an Evans Blue sieving technique. DHT-treated HAF-SS.5BN rats had significantly higher MAP compared to placebo-SS.5BN (128±6 vs. 104±1 mmHg, p<0.004). In contrast, HAF-CYP4A2-/- rats had no change in MAP compared to placebo-CYP4A2-/- controls (120±4 vs 118±3 mmHg, p=NS). Endogenous 20-HETE levels in renal microvessels of HAF-SS.5BN rats were significantly increased compared to Placebo-SS.5BN (2.27±0.91 vs. 0.32±0.037 pmol/mg, p<0.01). The 20-HETE levels were lower in CYP4A2-/- than SS.5BN but DHT in HAF-CYP4A2-/- had no effect on 20-HETE levels compared to Placebo- CYP4A2-/-. These results suggest that androgen-mediated upregulation of the expression of CYP4A2 and the production of 20-HETE in renal microvessels contribute to elevated BP in HAF rats. These data also suggest that methods to attenuate 20-HETE may provide a novel therapeutic to reduce BP in women with PCOS. Work supported by NIH RO1HL66072 and PO1HL51971.
Abstract P048: Blood-Pressure Associated Variants in Natriuretic Peptide Receptor C Affect Human Vascular Smooth Muscle Cells Proliferation and Calcium Flux in Response to Angiotensin II
Authors: Ren Meixia , Ng Fu Liang , Witkowska Kate , Baron Michael , Townsend-Nicholson Andrea , Xiao Qingzhong , Hobbs Adrian , Ye Shu , Caulfield Mark
Published: July 2021

Introduction:
A recent genome-wide association study revealed a significant association between variation at natriuretic peptide receptor C (NPR3) gene locus and blood pressure (BP).

Objective:
To functionally characterise the effect of BP- associated SNPs (single nucleotide polymorphisms) at the NPR3 gene locus in the context of BP regulatory pathways.

Methods:
A collection of primary human umbilical smooth muscle (HUASMCs) and endothelial (HUVECs) cells were genotyped for NPR3 gene SNP rs1173743, rs1173747, rs1173756 and rs1173771. Endogenous mRNA and protein expression levels were assessed by qRT-PCR and western blotting. Open chromatin regions were assayed using formaldehyde-assisted isolation of regulatory elements. Cell proliferation and migration were detected by cell counting and scratch assays. Angiotensin II (AngII)-induced Calcium flux was investigated using an intracellular fluorescent probe (Calcium 6).

Results:
The NPR3 gene risk allele (major allele) of intronic SNP rs1173747 was associated with lower endogenous mRNA (p<0.001) and protein levels in HUASMCs. This was consistent with its minor allele being located within an open chromatin state (p<0.05). Furthermore, cells carrying the major-allele of rs1173747 had increased proliferation (p<0.05) and Calcium flux in response to AngII stimulation (p<0.05). No difference in migration rates were detected. No such genotype-dependent characteristics were observed in HUVECs.

Conclusions:
This study has identified potential mechanisms for BP-associated SNPs in NPR3 gene locus to influence BP predominantly via effect on vascular smooth muscle cell behaviours
Abstract P052: Homocysteine Increases Macrophage-Derived Paraoxonase -1 Expression Independent of CD68
Authors: Chernyavskiy Ilya , Winchester Lee , Veeranki Sudhakar , Tyagi Suresh
Published: July 2021

Although atherosclerotic plaque rupture is the leading cause of myocardial infarction, the mechanisms are unclear. Macrophages burdened with oxidized LDL (oxLDL) become foam cells: hallmarks of plaque progression and instability. One of the main macrophage-specific receptors for oxLDL is CD68. Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated lactonase capable of retarding/inhibiting LDL oxidation. Elevated levels of homocysteine (Hcy), an amino acid homologue and independent cardiovascular risk factor, is metabolized by Pon1.

Given the literature connections of oxLDL, Pon1, Hcy, and macrophages to atherosclerosis, we hypothesized that Pon1 is produced by murine macrophages and its expression is increased by Hcy via CD68.

Murine J774a.1 macrophages were treated with LDL, oxLDL, Hcy, or oxLDL+Hcy. Also, separate treatment groups included macrophages that had CD68 silenced by CD68 siRNA transfection. Cell lysates were analyzed for CD68 and Pon1 expression via Western blotting.

Pon1 is present in macrophages. Hcy along with oxLDL significantly increases Pon1 (51%, 1.51 vs 0.97) expression compared to controls than oxLDL alone. Pon1 expression is significantly decreased (33%, 0.67 vs 1) with silencing of CD68. Pon1 expression is significantly decreased more with oxLDL (82% 0.17 vs 1) in presence of CD68 silencing but is significantly increased with oxLDL+Hcy (24% 1.24 vs 1). CD68 expression tends to increase more with oxLDL+Hcy than oxLDL alone when compared to control and the tendency follows with silencing of CD68. Our results conclude that Hcy increases macrophage-derived Pon1 expression independent of CD68.
Abstract P053: Nicotine-induced
Renal Inflammation
in Genetic Hypertension is
Not Dependent
on
Renal Sympathetic Nerve Activity
Authors: Harwani Sailesh , Chapleau Mark W , Meyerholz David , Abboud Francois
Published: July 2021

We recently demonstrated that cholinergic stimulation with nicotine in vivo leads to renal inflammation and premature development of hypertension in Spontaneously Hypertensive Rats (SHR), but not in Wistar Kyoto (WKY) controls. Nicotine can stimulate immune cells directly and influence inflammation indirectly by increasing sympathetic nerve activity (SNA). We hypothesized that increased renal SNA contributes to nicotine-induced renal inflammation in SHR. We tested this hypothesis by measuring the number of CD68+ macrophages in kidneys of prehypertensive SHR (3-5 weeks old, n=7) and age-matched WKY rats (n=3) after subcutaneous infusion of nicotine via osmotic mini-pump (625 mcg/kg/hr) for 24 hours. Each rat was subjected to unilateral renal nerve denervation (RND) and sham surgery on the contralateral kidney 1 week before implanting the mini-pump. RND failed to abrogate nicotine-induced renal inflammation, actually increasing CD68+ macrophage infiltration in renal tubulointerstitium of SHR (110 ± 3 vs. 89 ± 1 cells/unit area in denervated vs. contralateral intact kidneys, respectively, p<0.001). RND had no effect on the number of infiltrating CD68+ macrophages in the tubulointerstitium of WKY controls (83 ± 3 vs. 79 ± 2 cells/unit area in denervated vs. intact kidneys, respectively, p>0.05). Renal norepinephrine content was measured by ELISA to confirm RND. We conclude that nicotine-induced renal macrophage infiltration in SHR is not dependent on renal SNA, and speculate that renal SNA may be anti-inflammatory in this model.
Abstract P061: Selective Deletion of Intracellular Renin in the Brain Causes Hypertension and Elevated Sympathetic Nervous System Activity
Authors: Shinohara Keisuke , Weidemann Benjamin J , Folchert Matthew D , Liu Xuebo , Morgan Donald A , Rahmouni Kamal , Grobe Justin L , Sigmund Curt D
Published: July 2021

Renin expression is regulated by two distinct promoter-1st exon combinations that target renin either for secretion (exon 1a for secreted renin) or cytoplasmic retention (exon 1b for intracellular renin, icREN). We developed icREN knockout (KO) mice by selectively deleting exon 1b. icREN KO mice are essentially brain-specific knockouts of icREN because icREN is predominantly expressed in the brain. Notably, systolic blood pressure measured by telemetry was increased in icREN KO mice (130±2 mmHg, n=8 vs 122±2 mmHg in controls, n=7, P<0.01). The low- to high-frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability, a parameter of sympathetic nerve activity (SNA), was increased in icREN KO mice (KO: 1.24±0.21, n=7 vs control: 0.70±0.11, n=7, P<0.05). Body weight (BW) was normal in icREN KO mice compared to controls, but the BW gain and fat accumulation induced by high fat diet (HFD) were attenuated in male icREN KO mice (BW at 16 wks of HFD- KO: 36.8±1.2 g, n=8 vs control: 41.9±1.4 g, n=9; relative fat mass at 14 wks of HFD- KO: 27.7±1.7%, n=8 vs control: 34.4±2.3%, n=9, both P<0.05). The resting metabolic rate measured by respirometry was increased in icREN KO mice (0.156±0.005 kcal/h, n=46, P<0.05) vs controls (0.145±0.003 kcal/h, n=53), whereas food consumption and absorbed calories were not different. We previously reported that the brain renin-angiotensin system facilitates renal SNA (RSNA) response to acute intracerebroventricular (ICV) injection of leptin. Interestingly, the RSNA response to ICV leptin was greater in icREN KO mice (KO: 214±40 % baseline, n=5 vs control: 114±18 % baseline, n=10, P<0.01). AT1a receptor mRNA was upregulated in the paraventricular nucleus of icREN KO mice (P<0.05). Chronic ICV injection of losartan not only abolished the elevated blood pressure in icREN KO mice, but reduced it to below baseline in controls (systolic blood pressure, 111±3 mmHg in KO, n=5; 124±4 mmHg in controls, n=6). These data suggest that icREN deletion increases the activity of brain renin-angiotensin system and elevates blood pressure and metabolic rate through sympathetic activation. We conclude that this novel icREN isoform contributes to cardiovascular and metabolic control possibly as part of an inhibitory neural circuit.
Abstract P079: Impact of Combined Exercise Training in an Experimental Model of Hypertension Associated With Menopause
Authors: Shimojo Guilherme , Dias Danielle , Malfitano Christiane , Sanches Iris , Irigoyen Maria-Cláudia , De Angelis Katia
Published: July 2021

In this study we tested the hypothesis that the cardiovascular autonomic dysfunction plays an important role on the management of inflammation and oxidative stress, and that these dysfunctions may in turn be modulated by combined exercise training in an experimental model of hypertension and menopause. Female rats were divided into (n=7/group): control (C) and hypertensive (H), hypertensive ovariectomized (HO) and hypertensive ovariectomized undergoing combined (aerobic+resistance) training (THO). We observed an additional increase in HO group (176±4 mmHg) in relation to H group (165±3 mmHg). However, the THO group (155±3 mmHg) showed a reduction of arterial pressure associated with resting bradycardia. The HO group (50.78±4.61 mmHg2) presented an additional impairment in systolic arterial pressure variability when compared to C and H groups (23.69±0.45 and 34.09±2.37 mmHg2); this dysfunction was not observed in THO group (30.09±2.03 mmHg2). Additionally, an attenuation on vascular sympathetic modulation and an improvement in baroreflex sensitivity were found in the THO when compared to HO group. There was an increase in TNF-α in sedentary hypertensive groups (H and HO vs. C), which was not observed in THO group. Ovariectomy induced an additional increase in cardiac and renal oxidative stress, which were reduced in THO group. The THO group presented an increase in total antioxidant capacity when compared to the other groups. In conclusion, combined exercise training was able to reduce AP associated with improvement on cardiovascular autonomic control, probably reducing cardiac and renal inflammation and oxidative stress, in an experimental model of hypertension and menopause.
Abstract P120: Improving the Angiogenic Abilities of Mobilized Peripheral Blood Stem Cells Achieved by Priming with Activated Platelet Supernatant for Regenerative Cell Therapy
Authors: Choi Jae-Il , Hur Jin , Kang Jin-A , Yun Ji-Yeon , Lee Hwan , Nham Pniel , Hwang Injoo , Chae Cheongwhan , Kim Tae-Won , Kang Jeehoon , Yoon Chang-Hwan , Yang Han-Mo , Kwon Yoo-Wook , Cho Hyun-Jai , Park Young-Bae , Kim Hyo-Soo
Published: July 2021

Platelets play a critical role in hemostasis and also have ability to promote angiogenesis and tissue repair by secreting of numerous cytokine and making angiogenic condition. We investigated whether autologous ‘activated platelet supernatant (APS)’ has effect on enhancing pro-angiogenic potential of peripheral blood stem cells (PBSC) for stem cell-based therapy for ischemic diseases. Granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (mobPBSC) were isolated from healthy volunteers, while APS was collected from platelet rich plasma by thrombin activation. mobPBSCs were primed with APS (APS primed mobPBSCs) for 6 hours, and APS primed mobPBSCs characterized their angiogenic ability. For the safety analysis, we estimated the thrombogenicity of platelets in whole blood mixed with APS primed mobPBSCs by expression of glycoprotein IIb and IIIa on platelets. APS had a higher level of various cytokines, such as IL8, IL17, PDGF and VEGF than naïve platelet supernatants. And APS primed mobPBSCs had more expression of angiogenic factors, surface markers (i.e. CD34, CD31, and CXCR4) and integrins (integrin α5, β1 and β2) than Veh primed and Pre primed mobPBSC. Also APS primed mobPBSCs were polarized toward CD14++/CD16+ pro-angiogenic monocytes. And result in adhesion to endothelial cells and fibronectin which represents cell to cell and cell to extracellular matrix adhesion, respectively. The culture supernatant of APS-primed mobPBSCs contained high levels of IL8, IL10, IL17 and TNFα, and augmented proliferation and capillary network formation of HUVEC. In-vivo transplantation of APS-primed mobPBSC into athymic mice ischemic hindlimbs and Matrigel plugs elicited vessel differentiation and tissue repair. In thrombogenicity test, platelet activity increased after mixing whole blood with mobPBSC regardless of the priming agent. However, this was reduced by pretreatment of aspirin, which is an antiplatelet agent prescribed to patients with ischemic diseases. Our data demonstrate that mobPBSCs primed with APS improve angiogenic potential, and that can be adjunctive strategy to enhance the efficiency of stem cell therapy for ischemic diseases.
Abstract P127: Angiotensin II Priming Enhances Prostaglandin E2 Vasoconstrictor Effects
Authors: Kraemer Maria P , Lamb Fred , Breyer Richard M
Published: July 2021

Prostaglandins are key modulators of blood pressure and arterial tone. Prostaglandin E
2
(PGE
2
), is a prostanoid that has vasodepressor effects; however, under certain circumstances PGE
2
can induce vasopressor responses. Recent reports demonstrated that sub-threshold concentrations of vasoconstrictors augment PGE
2
-mediated constriction in rat femoral arteries. However, whether angiotensin II (Ang II) could affect PGE
2
-mediated contraction is not known. Using a wire myograph, we demonstrated that PGE
2
had no significant effect on mouse femoral arterial rings at doses up to 1 μM. However, priming of arterial rings with 1 nM Ang II potentiated PGE
2
-evoked constriction in a concentration dependent manner (Area Under the Curve, AUC
untreated
1.784 ± 0.353, AUC
Ang II
23.27± 9.820, P<0.05). We tested femoral arteries from EP1, EP2, and EP3 receptor knockout mice. Only the EP3-/- arteries were unable to respond to PGE
2
after Ang II priming (figure below). Pretreatment of arterial rings with 1 μM losartan, an angiotensin receptor antagonist, blocked PGE
2
-induced constrictor effects primed with Ang II (% of KCl, Ang II 21.72 ± 5.296, Ang II + losartan 3.025 ± 1.046, n=3). We have determined that re-addition of extracellular Ca
2+
to a Ca
2+
-free artery restores PGE
2
-induced contractions (n=5) and that the Rho-kinase inhibitor Y-27632 blocks contraction (n=3). Taken together these data are consistent with angiotensin AT1 and prostaglandin EP3 receptors mediating a synergistic Rho-kinase-dependent contractile response. We are continuing to investigate the relationship between Ang II and PGE
2
to determine the physiological relevance this may have in modulating blood pressure.
Abstract P147: Office Blood Pressure: An Inadequate Guide to the Diagnosis and Treatment of Hypertension
Authors: Cohn Jay , Duval Sue , Florea Natalia , Hoke Lynn , Duprez Daniel
Published: July 2021

Hypertension is a cardiovascular (CV) disease with high risk for CV morbid events (ME) that benefits from anti-hypertensive therapy. Resting blood pressure (BP) >140/90 mmHg serves as the diagnostic criterion for hypertension, and management has been aimed at BP reduction. Progression of CV disease in the absence of elevated blood pressure identifies individuals who might benefit from CV-protective therapy but are not currently being recognized as in need of treatment.

In 2017 asymptomatic individuals evaluated for early functional and structural CV abnormalities, 1534 not taking anti-hypertensive drugs were available to determine the relationship between office blood pressure and the severity of CV abnormalities, as defined by a 10-test non-invasive disease score (DS) of 0-20. Previous studies have documented the high predictive value of DS for future CVME.

The population was 53% male, average age 50±11 years, BP 122/77mmHg, LDL cholesterol 129±38 mg/dL, HDL 52±17mg/dL, triglycerides 109 mg/dL. DS was adjusted by eliminating the score for BP, but 9-test DS was still directly related to BP: 2.3 in those (n=550) <120/80 mmHg (Group I), 3.2 in those 120-129/80-85 mmHg (n=600) (Group II), 4.1 in those 130-139/85-89 mmHg (n=236) (Group III), and 5.7 in those 140+/90+mmHg (n=148) (Group IV). Nonetheless, DS of >6 indicative of high risk was present in 10% of Group I, 20% of Group II and 30% of Group III. BP was largely overlapping in individuals with no CV disease (DS 0-2), early disease (DS 3-5) and advanced disease (DS 6+).

Therefore, reliance on resting BP leaves many at-risk individuals undiagnosed and untreated for early CV disease likely to progress. The hypertensive state exists in the absence of elevated BP and should be recognized and treated to prevent CVME.
Abstract P154: Ultrasound Imaging for Serial Measurements of Venous Diameters in Rats
Authors: Krieger-Burke Teresa , Seitz Bridget M , Fink Gregory D , Watts Stephanie W
Published: July 2021

The purpose of our study was to investigate serial ultrasound imaging in rats as a means to quantify the diameters of splanchnic veins in real time and the effect of drugs on venous capacitance. A 21 MHz probe ( Vevo 2100 imaging system,Visual Sonics Inc.) was used to collect images containing the portal vein (PV) and the superior mesenteric vein (SMV) in anesthetized male Sprague-Dawley rats maintained at 37°C. Stable landmarks were established and we were able to repeatedly locate specific cross-sections of PV and SMV. When controlled for respiratory and cardiac cycles during measurements, respective diameters of these vessels remained within 0.75±0.15% and 0.2±0.10% of baseline (PV: 2.02±0.15 mm; SMV: 1.67±0.05 mm) when located and measured every 5 minutes over 45 minutes (n=3 rats). PV and SMV remained within 1.0±0.6% and 0.38±0.9% from baseline, respectively, when measured on separate days over 10 weeks in a preliminary study using 2 rats. The consistency of raw vessel measurements allowed these vessels to serve as their own control during subchronic pharmacologic interventions.

In a second study, the vasodilator sodium nitroprusside (2 mg/kg, i.v. bolus) was administered to anesthetized rats (n=3) following collection of baseline vessel measurements. PV and SMV diameters increased 37.23±2.4% and 29.77±8.8% from baseline by 30 minutes post drug administration while mean arterial pressure decreased 10.32±1.7 mmHg. Conversely, the administration of the venoconstrictor sarafotoxin (S6C) (5 ng/kg, i.v. bolus) to other anesthetized rats (n=3) decreased PV and SMV diameters 22.10±2.4% and 9.44±1.6% from baseline within 5 minutes, associated with an increase in mean arterial pressure of 12.85±3.2 mmHg.

Together these results support serial ultrasound imaging as a reliable technique to accurately measure acute and subchronic changes in the diameter of splanchnic veins concurrent with blood pressure changes in intact rats. The ability to follow rat abdominal vein diameters in real time will assist in determining the role of the venous circulation in blood pressure regulation.
Abstract P191: Increases in Cerebrospinal Fluid NaCl Concentration Excites Neurons of the Organum Vasculosum of the Lamina Terminalis to Elevate Sympathetic Outflow and Blood Pressure
Authors: Simmonds Sarah S , Nation Haly L , Stocker Sean D
Published: July 2021

Accumulating evidence suggests salt-sensitive hypertension is mediated partly by an increase in cerebrospinal fluid (CSF) NaCl concentration and elevation in sympathetic nerve activity (SNA). Increased NaCl concentration or osmolality is sensed by specialized neurons in the organum vasculosum of the lamina terminalis (OVLT). The present study investigated the contribution of these neurons to the SNA and arterial blood pressure (ABP) responses during acute increases in CSF NaCl concentrations. Male Sprague-Dawley rats were anesthetized with Inactin (120mg/kg, IV) and prepared for SNA and ABP recordings. Lateral ventricle infusion of 1M NaCl (5uL over 10 min) increased CSF [Na+] by 5±1 mM and elevated mean ABP (9±1 mmHg), lumbar SNA (125±3%) and adrenal SNA (121±5%) but decreased renal SNA (-9±1%, n=8) and did not alter splanchnic SNA (102±3%). Inhibition of the OVLT with injection of the GABAA agonist muscimol (2.5mM per 20nL, n=5) significantly attenuated the NaCl-induced increase in ABP (2±1 mmHg), lumbar SNA (102±1%), adrenal SNA (103±2%) and decrease in renal SNA (-3±1%). In vivo single-unit recordings demonstrate that lateral ventricular infusion of 1M NaCl (5uL per 10 min) significantly increased the firing rate in 75% (3/4) of OVLT neurons from 1.2±0.4Hz to 5.1±1.2Hz (P<0.05). Furthermore, direct injection of NaCl (100nL over 20s, n=3) into the OVLT produced dose-dependent increases in mean ABP (0.15M: 0±1mmHg; 0.5M: 2±1mmHg; 1.0M: 4±1mmHg; 2.0M: 7±1mmHg) and lumbar SNA (0.15M: 101±2%; 0.5M: 107±1%; 1.0M: 112±3%; 2.0M: 118±4%). Altogether, these findings suggest that increases in CSF NaCl concentrations excite OVLT neurons to elevate lumbar and adrenal SNA and ABP.
Abstract P199: Uric Acid Promotes Vascular Stiffness, Immune Inflammatory Response and Proteinuria in Western Diet Fed Mice
Authors: Aroor Annayya , Jia Guanghong , DeMarco Vincent G , Habibi Javad , Sun Zhe , Garro Mona , Martinez-Lemus Luis , Nistala Ravi , Whaley-Connell Adam , Meininger Gerald , Sowers James
Published: July 2021

Increased consumption of a diet high in fructose and fat (western diet, WD) is associated with an increase in cardiovascular disease (CVD) and kidney injury. In this regard, excess hepatic production of uric acid generated from excess fructose consumption is emerging as a risk factor for vascular stiffness, which underpins CVD and kidney injury. We hypothesized that a WD would increase uric acid levels and cardiovascular and renal xanthine oxidase (XO) activity and associated increased vascular stiffness and proteinuria. Furthermore, we proposed that inhibition of XO activity would prevent arterial stiffening and reduce proteinuria in a clinically relevant model of WD-induced CVD and renal injury. Four week-old C57BL6/J male mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without allopurinol (125mg/L), a potent XO inhibitor for 16 weeks. XO inhibition significantly attenuated WD-induced increases in plasma and urine uric acid levels and aortic XO activity (WD, 0.225 + 0.031 mU/mL WD + allopurinol, 0.097+ 0.026mU/mL, P<0.05), as well as proteinuria (WD, 20.92 + 2.66 mg/ mg creatinine, WD + allopurinol, 13.48 + 1.56 mg/mg creatinine, P<0.05). XO inhibition had no effect on increases in body weight, fat mass, and HOMA-IR promoted by the WD. Blood pressure was not different between any of the groups. Stiffness of aortic endothelial cells, extracellular matrix and vascular smooth muscle cells, as determined by atomic force microscopy, was significantly increased in WD mice and this was prevented by XO inhibition. WD induced a significant macrophage pro-inflammatory response in aorta that was significantly suppressed by XO inhibition. Collectively, these findings support the notion that increased XO activity in the vasculature and kidney and increased hepatic production of uric acid secondary to consumption of a WD promotes vascular stiffness, vascular inflammation and a maladaptive immune response that lead to vascular stiffness and kidney injury.
Abstract P207: Role of (Pro)Renin Receptor in the Pathogenesis of Colon Cancer
Authors: Nishiyama Akira , Wang Juan , Yachida Shinichi , Nguyen Genevieve , Hirose Takuo , Shibayama Yuki
Published: July 2021

(Pro)renin receptor ((P)RR) is a component of the Wnt receptor complex (Science, 2010). We have recently demonstrated that (P)RR plays an important role in the tumorigenesis of pancreatic ductal adenocarcinoma via the activation of Wnt/β-catenin signaling pathway (Shibayama et al. Sci Rep. 2015). Since the patients with colon cancer often show aberrantly activated Wnt/β-catenin-dependent signaling pathway by the mutations of its components, we investigated the possible role of (P)RR and Wnt/β-catenin signaling pathway in carcinogenesis of colon cancer. Real-time PCR was used for measuring mRNA levels of (P)RR. Protein levels of (P)RR was determined by Western blotting and immunohistochemistry. Activated β-catenin levels were determined by Western blotting. Cell proliferative ability was evaluated by counting the cell number in cultured colon cancer cell lines, HCT116 and DLD-1 cells. As compared to normal colon tissues (n=6), mRNA and protein levels of (P)RR were increased by 2.6- and 2.2-fold, respectively, in colon cancer tissues (n=9), which were associated with increased activated β-catenin levels (by 2.8-fold, P<0.05). However, plasma soluble (P)RR levels were not changed in patients with colon cancer (n=9). (P)RR and activated β-catenin levels were also increased in HCT116 (by 2.2- and 2.7-fold, n=5, respectively) and DLD-1 cells (by 1.9- and 2.8-fold, n=5, respectively). In these cells, inhibiting (P)RR with an siRNA attenuated the activity of β-catenin and reduced the proliferative abilities (n=5, P<0.05, respectively). These data suggest that (P)RR contributes to the tumorigenesis of colon cancer through the activation of Wnt/β-catenin signaling pathway.
Abstract P210: Association of Perceived Stress and Aldosterone and Adiponectin Levels in a Rural Cohort of African Americans
Authors: Pointer Mildred A , Sanchez Jesus , Hicks Kianda , Wells Candace , Bridges Lakeesha , Bolin David , Greene Natasha
Published: July 2021

African Americans (AA) have the highest prevalent rates of ESRD than any other group in this country. AA males have the highest risk with more than a two-fold greater risk of kidney injury compared to their female counterparts. The underlying pathophysiology that predisposes AA males to renal injury remains to be definitively determined. However, recent findings from our laboratory have shown that stress may exacerbate the renal injury associated with hypertension. Using an animal model of nitric oxide deficiency hypertension we showed that renal injury associated with this model of hypertension was increased following four weeks of intermittent stress (Pointer et al. 2012). The current study sought to determine whether we could find a similar association of stress and renal injury in a population prone to renal injury. To achieve this we recruited African Americans (11 males; 36 females) attending a health fair. We administered a perceived stress scale; measured resting blood pressure; and collected a blood sample for cortisol, adiponectin, and aldosterone measurement. Aldosterone (ALDO) is a hormone released from the adrenal gland in response to stress. ALDO levels have been linked to increased cardiovascular injury such as acute myocardial infarction and kidney disease. Adiponectin is an adipokine and appears to have protective actions in metabolic disease. In this sampling we found that non-normotensive (blood pressure above 120/80 mmHg) AA females had similar PSS scores as the males (24 ± 2 vs. 21 ± 2) and similar plasma cortisol levels (9.4 ± 0.5 vs. 9.8 ± 1μg/dL, respectively). However, in males cortisol was positively associated with ALDO (p=0.032) and negatively associated with adiponectin (p<0.03). There was no significant association of cortisol with ALDO and adiponectin in females. These findings suggest that AA males may be predisposed to renal injury as a consequence of stress leading to 1) increased injury promoting (ALDO) and 2) decreased disease protective (adiponectin) agents. Further studies are needed to confirm these findings in a larger sampling.
Abstract P226: Biochemical Properties of the N-terminally Palmitoylated Adrenomedullin
Authors: Tokashiki Mariko , Kubo Keishi , Kuwasako Kenji , Nagata Sayaka , Kato Johji , Kitamura Kazuo
Published: July 2021

Purpose:
Adrenomedullin (AM) is a potent vasodilator peptide having pleiotropic effects including cardiovascular protection and angiogenesis. Because of these beneficial effects, AM appears to be a promising therapeutic tool for human diseases such as myocardial infarction or peripheral artery disease, while intravenous injection of AM stimulates sympathetic nerve activity due to the short-acting potent vasodilation resulting in increased heart rate and renin secretion. To lessen those acute unfavorable actions, we conjugated human adrenomedullin N-terminally with palmitic acid, and examined biological effects of palmitoylated AM in the present study.

Methods:
Synthesized human AM peptide was conjugated with palmitic acid, and then palmitoylation AM was purified by HPLC. Biological effects in vitro stimulating intracellular cAMP, a major second messenger of AM, were examined using cultured human embryonic (HEK)-239 cells stably expressing a specific AM receptor. Blood pressure-lowering effects in vivo were tested by intravenous injections of palmitoylated AM or native AM peptides into anesthetized rats. Plasma disappearance curve of peptides were evaluated by the two compartment model.

Results:
Palmitoylated AM stimulated intracellular accumulation of cAMP in cultured HEK-293 cells, as did native human AM peptide, in a dose-dependent manner.

pEC50 of palmitoylatted AM was lower than natibe AM (8.49±0.12 vs. 9.17±0.12, mean±SEM, P<0.05), but no difference was noted in the maximum response of cAMP (579.9±24.5 vs. 667.2±24.5 pmol/well) . The first and second phases of plasma half-lives of native AM were 0.276 sec and 780 sec, while those of palmitoylated AM were 1.15 min and 599 min, respectively. Both half-lives of the palmitoylated peptide were significantly prolonged, as compared with the native peptide (P<0.05).

Conclusions:
N-terminally palmitoylated AM stimulated cAMP production in vitro, showing smaller acute hypotensive action and a prolonged plasma half-life in comparison of native AM peptide in vivo. The present results suggest a possibility for palmitoylated AM as a therapeutic tool with lessened unfavorable effect of acute hypotensin of native AM.
Abstract P249: Central Nervous System Control of Plasma Aldosterone and Endogenous Ouabain in Angiotensin II-Salt Hypertension
Authors: Lu Jiao , Wang Hong-Wei , Keshtkar-Jahromi Marzieh , Hamlyn John M , Leenen Frans H
Published: July 2021

High salt intake markedly enhances hypertension induced by Ang II. We evaluated peripheral mechanisms which may amplify central mechanisms activated by Ang II-salt. In the 1
st
exp, Wistar rats were sc infused with Ang II at low dose of 150 ng/kg/min together with 2% high salt diet for 14 days. In the 2
nd
exp, MR blockers (eplerenone, spironolactone), ENaC blocker (benzamil), AT
1
R blocker (losartan) or vehicles (Veh) were icv infused combined with Ang II-salt. BP was recorded by telemetry. Plasma corticosterone (Cor), aldosterone (aldo) and endogenous ouabain (EO) were measured by RIA. Gene expression was assessed by real-time qPCR. Ang II alone caused a small increase in MAP (112±1 vs. 99±1 mmHg), but BP was markedly increased by Ang II-salt (152±4 mmHg, P<0.05 vs. others). BP increases to Ang II-salt were largely inhibited by central infusion of MR blockers, benzamil or losartan. Only Ang II together with salt increased plasma aldo, Cor and EO. In the adrenal cortex, both Ang II alone and Ang II-salt increased CYP11B2 expression but neither affected CYP11B1, Hsd3b1 or AT
1
R mRNA expression. Central blockades significantly (p<0.05) lowered plasma aldo and EO in rats on Ang II-salt. Central blockades had no effect on Hsd3b1, CYP11B1 and AT
1
R mRNA but markedly decreased Ang II-salt induced CYP11B2 expression in the adrenal cortex. Together, these results suggest that in Ang II-salt hypertension, Ang II-AT
1
R signaling and MR-ENaC pathway in the brain increase plasma aldo and EO, which may amplify BP responses to central mechanisms and contribute to severe hypertension by Ang II-salt vs Ang II alone.
Abstract P610: Alterations in 20-HETE Production Contribute to End Organ Damage in Dahl S Rats
Authors: Fan Fan , Wu Wenjie , Murphy Sydney
Published: July 2021

It is well documented that a sexual dimorphism exists in the regulation of blood pressure in both the human population as well as experimental animal models, such that males have higher blood pressures than females of the same age. While there is a clear disparity in the development of hypertension and progression of renal injury in many rodent models, evidence of a sex difference is lacking in the Dahl S rat. While the current reports present conflicting data, we hypothesize that alterations in CYP450 expression and 20-HETE production contributes to the relative resistance of female Dahl S rats to target organ damage compared to males. Consistent to what we have previously reported, the time course for the development of proteinuria and renal injury were significantly reduced in female Dahl SSJr rats challenged with a high salt diet relative to male rats. In addition, renal cortical 20-HETE production was elevated in female (120.8±4.2 pmol/min/mg) versus male rats (45.1±11.78 pmol/min/mg), while no difference was noted in the outer medulla (19.6±1.8 vs 17.15± 3.9 pmol/min/mg). Introgression of the CYP4A1 gene into the Dahl S genetic background, resulted in significant elevations in 20-HETE production both on low salt and high salt diets. Furthermore the rise in mean arterial pressure was attenuated in CYP4A1 overexpressing rats in both sexes (Δ19mmHg in CYP females vs 61mmg in Dahl S females; 20mmHg in CYP males vs 50mmHg in Dahl S males). Moreover, the degree of glomerular injury was reduced in CYP rats, both male and female, compared to Dahl S rats in response to a high salt diet. Therefore, increases in the CYP gene expression and 20-HETE production prevent the rise in mean arterial pressure and kidney injury in male Dahl S rats. AHA 14SDG20160020
Abstract P636: Sex Differences in Cardioprotective AT2R Expression in Diabetic Rats and Its Correlation with Myocardial Damage
Authors: Lum-Naihe Kelly , Mahmood Abuzar , Bajwa Jamal , Emter Craig A , Pulakat Lakshmi
Published: July 2021

Diabetes mellitus (DM) is an independent risk factor for cardiovascular disease (CVD). Healthy, young women are protected from CVD, while diabetic women are more susceptible to CVD compared to age-matched diabetic men and non-diabetic women. Underlying mechanisms for this sex difference in CVD are not fully elucidated. The angiotensin II type 2 receptor (AT2R) is a member of the protective, vasodilative arm of the renin angiotensin system. The Agtr2 gene that codes for AT2R is X-linked, and increased Agtr2 expression is reported in female vasculature of rodent models. We hypothesized that a sex difference might exist in DM-associated regulation of cardiac AT2R expression. To test this, we used hyperglycemic, male and female Zucker diabetic fatty (ZDF) rats and age- and sex-matched normoglycemic Zucker lean (ZL) rats. The male ZDF (ZDF-M) rat is an established model of type 2 DM. We have reported previously that hyperglycemic, female ZDF (ZDF-F) rats had the highest body fat and lowest lean muscle mass compared to male and female lean rats (ZL-M, ZL-F) and ZDF-M. Cardiac Agtr2 expression was measured by qRT-PCR at 5-months, cardiac function by echocardiography was compared at 3- and 5-months, and histopathology of cardiac tissue was assessed at 5-months. ZL-F had a nearly 2-fold increase of Agtr2 compared to ZL-M (p<0.01). Relative to lean controls, ZDF-M had no significant change in Agtr2, while ZDF-F exhibited ~60% suppression (Rq=0.42) of Agtr2 (p<0.001). Echocardiography data revealed evidence of compensated systolic function in all groups since fractional shortening was >50% at both ages, while heart rate and stroke volume were similar. However, diastolic dysfunction was observed in both ZDF-F and ZDF-M, relative to their lean counterparts, due to increased isovolumic relaxation time and decreased early:late ventricular filling ratio (E/A). ZDF-F exhibited the highest cardiomyocyte hypertrophy (≥35% over ZL-F, ZL-M and ZDF-M). Both ZDF and ZDF-M showed mitochondrial clustering and disrupted spatial orientation of mitochondria relative to the sarcomere (assessed by TEM). Based on our results, we propose that myocardial remodeling, diastolic dysfunction and loss of cardioprotective AT2R may underlie greater susceptibility of diabetic females to CVD.
Abstract 003: Role of Transient Receptor Potential Ankyrin 1 in Monocyte Activation and Hypertensive Kidney Injury
Authors: Ma Shuangtao , He Kecheng , Wei Shujie , Wang Peijian , Yang Yongjian , Wang Donna H
Published: July 2021

It has been shown that monocyte activation and infiltration occur during the development of hypertensive kidney disease, a condition newly recognized as an inflammatory disorder. Indeed, monocytosis is linked to chronic kidney disease. Transient receptor potential ankyrin 1 (TRPA1), a Ca
2+
-permeable non-selective cation channel, has been shown to be an oxidative stress sensor that regulates neurogenic inflammation due to its abundant expression in sensory nerves. With the use of qPCR and immunostaining, we identified that the TRPA1 is also expressed in human and murine monocytes. The gene expression of TRPA1 is decreased in peripheral monocytes in hypertensive patients compared to normotensive subjects (-6-fold, p<0.05) and in kidney tissues in angiotensin II (1000ng/kg/min, s.c., 14 days)-induced hypertensive compared to control mice (-9-fold, p<0.01). The differentiation of human monocytic leukemia cells (THP-1) into macrophages induced by phorbol 12-myristate 13-acetate (PMA, 100nM, 48h) was accompanied with decreases in TRPA1 expression (-8-fold, p<0.05) and increases in pro-inflammatory cytokines, including interleukin (IL)-1β (+705-fold, p<0.01), monocyte chemotactic protein (MCP)-1 (+16-fold, p<0.01), and CD36 (+7-fold, p<0.01). Treatment with supercinnamaldehyde (SCA), a TRPA1 agonist, decreased the expression of IL-1β (-12-fold, p<0.01), MCP-1 (-3.5-fold, p<0.01), and CD36 (-2-fold, p<0.05) in THP-1-derived macrophages, whereas these effects were abolished by pre-treatment with the selective TRPA1 antagonist, HC-030031 (HC). Additionally, HC promoted the proliferation of THP-1 cells while SCA suppressed THP-1 cell growth and variability. SCA induced the apoptosis of THP-1 cells (Annexin V positive cells: 8.5±1.4%
vs.
30.1±2.4%, p<0.01), which can be blocked by HC (30.1±2.4%
vs.
15.6±1.9%, p<0.01). These results show that TRPA1 activation inhibits monocyte activation and differentiation, and that suppressed expression of TRPA1 occurs in monocytes and kidneys in hypertensive patients and animals, respectively. These findings indicate that impaired expression and function of TRPA1 may contribute to monocytosis and pro-inflammatory state during hypertension, leading to hypertension related kidney injury.
Abstract 005: Loss of Interleukin 21 Protects Against Hypertension and Associated Inflammation and End Organ Damage
Authors: Dale Bethany L , Pandey Arvind , Laroumanie Fanny , Itani Hana , Xiao Liang , Norlander Allison , Foss Jason , Algood Holly , Madhur Meena
Published: July 2021

T cell derived cytokines such as interferon gamma (IFNγ) and interleukin 17A (IL17A) are upregulated by and can promote angiotensin II-induced hypertension and end-organ damage. Interleukin 21 (IL21) is produced primarily by T follicular helper (Tfh) cells and has the potential to promote IL17A and IFNγ production from T effector cells while inhibiting T regulatory cells. IL21 is also a potent activator of germinal center (GC) B cells.
Thus, we hypothesize that IL21 promotes hypertension and hypertensive end-organ damage through its effect on T cell polarization and GC B cell activation.
The data indicate that indeed IL21
-/-
mice exhibit a 30 mmHg reduction in blood pressure in response to 4 weeks of angiotensin II (Ang II) infusion compared to age-matched wild type (WT) mice (p=0.0275). IL21 mRNA expression increases 1.5 fold in splenic T cells in response to Ang II infusion (p=0.015). Moreover, Tfh cells and GC B cells are increased in the aortas of WT mice after Ang II infusion (p=0.0136 and p=0.0067, respectively). Further, IL17A production from splenic CD4+ T cells and IFNγ production from splenic CD8+ T cells was reduced in IL21
-/-
mice compared to WT mice (p=0.0025 and p=0.0237, respectively) following Ang II infusion. Renal function was assessed by measuring albuminuria. WT mice developed 2-fold more albuminuria compared to IL21
-/-
mice in response to Ang II infusion (p=0.0349). Lastly, Ang II infusion impaired endothelium-dependent relaxation to acetylcholine in mesenteric vessels from WT mice (52.9% vs 17.5%; p=<0.0001). IL21
-/-
mice demonstrated moderately impaired endothelium-dependent relaxation at baseline (34.9%) but interestingly, there was no further impairment in these vessels following Ang II infusion. Taken together, these studies suggest that IL21 and the Tfh cell–GC B cell axis may play a key role in hypertension and hypertensive end-organ damage.
Abstract 023: Inducible Deletion of Adipocyte Prorenin Receptor Reverses Obesity Related Hypertension
Authors: Gatineau Eva , Cohn Dianne , Gong Ming , Yiannikouris Frédérique
Published: July 2021

Obesity contributes to approximatively 2.5 million deaths every year and is associated with life threatening conditions including hypertension. Recently, we found that constitutive deletion of adipocyte (pro)renin-receptor (PRR) prevented high-fat diet-induced obesity through a drastic decrease in fat mass. However, adipocyte PRR deficient mice were characterized by a fatty liver and by an elevated systolic blood pressure (SBP), classic features of models of lipodystrophy. The purpose of this study was to investigate whether the temporally-controlled deletion of adipocyte PRR in obese mice reverses obesity related hypertension. After 18 weeks of high fat diet, inducible adipocyte-PRR deficient (

PRR
ERT

) and control (
PRR
fl/Y
) male mice (n=7-11 mice/ group) were injected intraperitoneally with tamoxifen (TMX) for 5 consecutive days. Body weight, body composition and blood pressure, measured by radiotelemetry in a subgroup of mice (n=2-4 mice/ group), were recorded before and after TMX injection. The inducible deletion of adipocyte PRR in

PRR
ERT

mice decreased significantly body weights (

PRR
fl/fl

, 46.6 ± 1.3 g;

PRR
ERT

, 42.1 ± 1.4 g, P<0.05) and fat mass (

PRR
fl/fl

, 15.8 ± 1.0 g;

PRR
ERT

, 8.1 ± 0.7 g, P<0.05) compared to control mice. PPARγ, FABP4 and FAS mRNA levels were significantly decreased by 68% (6.8 out 10), 80% (8 out 10) and 68% (6.8 out 10) respectively in white adipose tissues of

PRR
ERT

mice suggesting that PRR positively regulated adipogenesis and lipid metabolism in adipose tissue. In addition, the inducible deletion of adipocyte PRR in

PRR
ERT

mice decreased significantly SBP compared to control mice (

PRR
fl/fl

, -4.3 ± 3.2 g;

PRR
ERT

, -10.2 ± 2.4 g, P<0.05). Interestingly, adipocyte angiotensinogen mRNA abundance was significantly decreased in adipose tissue of

PRR
ERT

mice fed a standard diet suggesting that the decrease in blood pressure might be mediated by a local renin angiotensin system (RAS). The measurement of local (liver, kidney, adipose tissue and brain) and systemic RAS in HF-fed mice is under investigation. Taken together, our results highlight a new signaling pathway in which PRR regulates adipogenesis, lipid metabolism and blood pressure. PRR could represent a new potential therapeutic target for obesity and hypertension.