bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 97: Histone Deacetylase Inhibition Prevents Cardiac Remodeling in Mice Carrying the Disruption of Guanylyl Cyclase/natriuretic Peptide Receptor-A Gene
Authors: Subramanian Umadevi , Kumar Prerna , Pandey Kailash N
Published: July 2021

Regulation of gene expression plays an obligatorily role in the modification of chromatin structure that dynamically attenuates cardiac hypertrophy. In order to culminate the role of epigenetic regulators in the heart tissue, the current study was undertaken to elucidate the effect of histone deacetylase (HDAC) inhibitor, sodium butyrate (SB) in cardiac remodeling process of
Npr1
(coding for GC-A/NPRA) gene-targeted mice. Wild type (
Npr1
+/+
, 2-copy), gene-disrupted (
Npr1
+/-
, 1-copy), and gene-duplicated (
Npr1
++/+-
, 3-copy) mice were administered intraperitoneally with SB (0.5 mg/kg/day) for 2 weeks (8 mice/group). Mice with gene-disruption (
Npr1
+/-
, 1-copy) exhibited the increase in cardiac hypertrophy, heart weight/body weight (HW/BW) ratio (6.9 ± 0.2), and systolic blood pressure (SBP, 121.5 ± 4 mmHg) compared with 2-copy (HW/BW, 5.1 ± 0.2; SBP; 100.9 ± 6 mmHg) and 3-copy (HW/BW, 4.7 ± 0.1; SBP, 89.4 ± 2 mmHg) mice. In addition, an increased activity of HDAC (3-fold, p<0.01) and decreased activity of histone acetyltransferases (HAT) (2.5-fold, p<0.01) were found in untreated 1-copy mice hearts. Whereas, 1-copy mice treated with SB showed reduced HW/BW ratio (5.7 ± 0.3), SBP (SB, 101.2 ± 2), HDAC activity (p<0.01) and improved HAT activity (3-fold, p<0.001). Also, a stimulatory effect on HAT activity was observed in SB treated 2-copy (30%, p<0.01) and 3-copy (50%, p<0.01) mice. Furthermore,
Npr1
+/-
mice showed a significant increase in the expression of hypertrophic marker genes such as β-myosin heavy chain (β-MHC, 2-fold), α-skeletal actin (α-SK, 2-fold), c-fos (2.5-fold), and c-jun (3-fold) compared to untreated 2-copy and 3-copy mice. A substantial attenuation in the expression of hypertrophic markers (β-MHC, 2.5-fold; α-SK, 2.4-fold) and matrix genes (MMP-2, p<0.01; MMP-9, p<0.01) was found in SB-treated
Npr1
+/-
mice. The basal expression levels of matrix proteins were also significantly reduced in 2-copy and 3-copy mice hearts. The results show that sodium butyrate-dependent inhibition of HDAC activity attenuates cardiac hypertrophy and fibrosis by improving HAT activity suggesting that chromatin modification can prevent cardiac remodeling process in a
Npr1
gene-dose-dependent manner.
Abstract 001: Axl Expression by CD4+ T Lymphocytes Promotes Salt-Dependent Hypertension
Authors: Wadosky Kristine M , Batchu Sri N , Hughson Angie , Donlon Kathy , Morrell Craig N , Fowell Deborah J , Korshunov Vyacheslav A
Published: July 2021

Introduction:
Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl.

Methods and Results:
We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p<0.05) in Axl-/- (396±23) compared to Axl+/+ (512±42) under Th1-priming. However, Axl had no effect on interleukin 4 (IL-4, ng/mL) production under Th2 polarizing conditions. Intracellular staining of the Th1/Th2 cells with IFN-γ and IL-4 antibodies by flow cytometry confirmed expression of cytokines in culture media. Complete blood counts showed that Axl-/- mice had significantly lower white blood cells due to decreased numbers of lymphocytes (4.5±0.7x10
9
) compared to Axl+/+ mice (7.8±0.7x10
9
). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice.

Conclusions:
Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.
Abstract 003: Cholinergic Expansion of an Inflammatory Macrophage Population in the Pre-Hypertensive Spontaneously Hypertensive Rat
Authors: Harwani Sailesh , Chapleau Mark W , Sutterwala Fayyaz , Ballas Zuhair , Meyerholz David , Abboud Francois M
Published: July 2021

Our laboratory previously identified an abnormally elevated CD161a+ immune cell population in splenocytes of the pre-hypertensive Spontaneously Hypertensive Rat (SHR) that was abnormally expanded following cholinergic activation with nicotine . In the present study, we tested the hypothesis that the expanded CD161a+ cell population represents an activated monocyte/macrophage population and are present in the bone marrow (BM). We isolated cells from the spleen and BM of pre-hypertensive (4-5 week old) SHR (n=3) and age-matched normotensive Wistar Kyoto (WKY, n=3) rats. Isolated cells were stained with a fluorochrome conjugated anti-rat CD161a monoclonal antibody and analyzed by flow cytometry. CD161a+ cells were more prevalent in splenocytes and BM of the pre-hypertensive SHR, compared to WKY (8.7 ± 1.4% vs 1.2 ± 0.6% and 12.6 ± 1.8% vs 1.7 ± 0.8%, respectively, p<0.001). BM cells and splenocytes were cultured for 36-48 hours in the presence or absence of nicotine (10μM) and then stained with fluorochrome conjugated anti-rat CD161a and CD68 antibodies. CD68 is a well accepted pan-macrophage marker that is up-regulated on activated monocytes/macrophages. The CD161+/CD68+ macrophages were comparable between the WKY and SHR in freshly isolated cells from the spleen (0.3 ± 0.2% vs 0.9 ± 0.5%, respectively) and BM (0.6 ± 0.4% vs 0.7 ± 0.2%, respectively). However, nicotine strongly expanded the CD161a+/CD68+ macrophage population in the BM of the SHR (1.1 ± 0.15% to 11.9 ± 3.45%, p<0.001) and WKY (1.7 ± 0.17% to 11.0 ± 1.9%, p<0.001). In contrast, the response of splenocytes to nicotine was strong in SHR (1.8 ± 0.40% to 10.7 ± 1.1%, p<0.001), but not significant in WKY (1.9 ± 0.4% to 3.1 ± 0.6%, p>0.05). Nicotine had no effect on the CD161a+/CD68- cell population in either splenocytes or BM cells of the SHR or WKY. Thus, an activated monocyte/macrophage population (CD161a+/CD68+) is expanded by cholinergic activation in both the BM and spleen of SHR, but only in the BM and not spleen of the WKY. Since, the pro-inflammatory nicotinic response present in the BM of the WKY is abrogated in the peripheral spleen, we conclude that the retention of this response in the SHR may contribute to the development of hypertension.
Abstract 008: Novel Pathological Diagnosis Between Aldosterone Producing Adenoma And Idiopathic Hyperaldosteronism
Authors: Satoh Fumitoshi , Ono Yoshikiyo , Morimoto Ryo , Kudo Masataka , Iwakura Yoshitsugu , Omata Kei , Seiji Kazumasa , Takase Kei , Nakamura Yasuhiro , Doi Masao , Okamura Hitoshi , Gomez-Sanchez Celso , Sasano Hironobu , Ito Sadayoshi
Published: July 2021

The expressions of steroidogenic enzymes to produce aldosterone like CYP11B1, CYP11B2, 3BHSD1 and 3BHSD2 remain to be clarified to confirm pathological subclassification between aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA). Furthermore, we examined the correlation between APA tumor size and the status of intratumoral steroidogenic enzymes involved in aldosterone biosynthesis using immunohistochemistry. Surgically proven forty APA patients and ten IHA patients were retrospectively studied. Multi-detector computed tomography, AVS, and laparoscopic adrenalectomy were performed in all of the patients studied. The tumor area of APA at the maximum diameter of the sections was precisely measured by ImageJ software. The status of steroidogenic enzymes was immunohistochemically analyzed using monoclonal antibodies for CYP11B1, CYP11B2, 3BHSD1 and 3BHSD2, and the findings were evaluated according to the H-score system, based on both the number of immunopositive cells and relative immunointensity. Adrenal masses were not detected by computed tomography in 20 APA patients.In all of 10 IHA patients, hyperplastic zona glomerulosa was accompanied by an expression of HSD3B1. In contrast, tumor cells in all 40 APA patients were not immunopositive to HSD3B1, but strongly and dominanty expressed HSD3B2. Perhaps, due to compensatory responses to excess aldosterone, APA had an adjacent zona glomerulosa whose immunoreactivities to HSD3B1 and HSD3B2 were profoundly reduced in all 40 APA patients. Maximum tumor area obtained in the specimens was significantly correlated with preoperative plasma aldosterone concentration, urinary aldosterone excretion, the H-score of CYP11B1, and was inversely correlated with the H-score of CYP11B2. These results demonstrated that small adenomas could produce sufficient aldosterone to cause clinically overt primary aldosteronism because of the significantly higher CYP11B2 expression per tumor area. Monoclonal antibodies against HSD3B1 and HSD3B2 could be useful for immunohistochemical differentiation between APA and IHA. In addition, the relatively higher CYP11B2 expression per area in smaller APA could clinically cause PA despite their CT-undetectable tumor size.
Abstract 015: Regulation of Resting Metabolism by the Angiotensin AT
2
Receptor
Authors: Littlejohn Nicole K , Weidemann Benjamin J , Pearson Nicole A , Markan Kathleen R , Potthoff Matthew J , Sigmund Curt D , Grobe Justin L
Published: July 2021

The renin-angiotensin system (RAS) positively correlates with obesity, and contributes to energy homeostasis through opposing actions in the brain and adipose. We hypothesize that site- and receptor-specific modulation may represent a novel therapeutic target for obesity. Transgenic “sRA” mice exhibit brain-specific RAS hyperactivity through expression of human renin in neurons (synapsin promoter) and human angiotensinogen via its own promoter. Previously we documented that sRA mice exhibit a suppressed circulating RAS, and an elevated resting metabolic rate (RMR) that is sensitive to replacement of circulating angiotensin II or the AT
2
receptor (AT
2
R) agonist, CGP-42112a (CGP, 100 ng/kg/min, s.c.). sRA mice consume more food than littermate controls (con n=7, 12.98±0.65 vs sRA n=8, 15.41±0.70 g/d, P<0.05), but because of a major suppression of digestive efficiency (con 77.6±2.3 vs sRA 59.3±4.7 % consumed, P<0.05), sRA mice absorb a normal number of calories (con 10.04±0.50 vs sRA 9.07±0.76 kcal/d). Chronic CGP had no effect on total daily caloric absorption (con+CGP n=7, 9.83±0.95 vs sRA+CGP n=5, 9.32±0.80 kcal/d); however CGP appears to disproportionately increase weight gain in sRA mice (vehicle +0.33±0.49 vs CGP +1.54±0.47 g/8 wks, P=0.27) compared to control mice (vehicle +2.12±1.00 vs CGP +2.47±0.73 g/8 wks, P=0.73), consistent with a suppression of energy expenditure by CGP. Given the increased RMR and core temperature in sRA mice, we next examined the expression of uncoupling protein-1 (UCP1) content of thermogenic adipose tissues by Western blot (all n=3, vs perilipin). Interscapular brown adipose tissue UCP1 was unchanged in sRA mice (94±6%), and weakly suppressed by CGP treatment (84±4% of sRA). Inguinal adipose UCP1 was increased in sRA mice (198±13%), and this was suppressed by CGP treatment (72±9% of sRA). UCP1 mRNA levels paralleled protein in both fat types. Cultured adipocytes from 4 day old AT
2
-deficient mice exhibited increased UCP1 protein vs littermate controls (190%). Further, CGP (10 nM) reduced UCP1 in control adipocytes (by 30%). These data support a suppressive action of AT
2
R upon RMR most likely through UCP1. Inguinal adipose AT
2
R may therefore contribute to obesity through suppression of RMR.
Abstract 021: Haplotype-Dependent Differential Regulation Of The Human AT1R Gene Is Exacerbated By Age: Effects On Tissue Inflammatory And Redox Milieu
Authors: Rana Anita , Jain Sudhir , Puri Nitin , Mopidevi Brahmaraju , Kaw Meenakshi , Kumar Ashok
Published: July 2021

Age-associated inflammation and redox imbalance underlie etiopathogenesis of cardiovascular-renal diseases, including hypertension and end organ damage. Angiotensin II (Ang II), via activation of the AT1R, contributes to the development and progression of these pathophysiologies. We have identified two haplotype blocks of single nucleotide polymorphisms (SNPs) in the hAT
1
R gene: haplotype II (Hap II: -810A, -713G, -214C, -153G) and I (Hap I: -810T, -713T, -214A, -153A). In clinical studies, Hap I is linked to human hypertension. This study examines haplotype-dependent and age-associated transcriptional regulation of the hAT1R gene. In this regard, we have engineered transgenic (TG) mice with either haplotype of the hAT1R gene using a 166-kb bacterial artificial chromosome. ChIP assay shows increased RNA-Pol II binding (~1.6 fold higher) to the chromatin extracts from renal tissues of adult (4-6 months) male Hap I-TG mice with increased hAT1R expression (~6 fold higher). This was accompanied by higher baseline blood pressure in Hap I-TG mice (Hap I- 129±3 vs. Hap II- 116±4, p<0.05). Next, we examined the effects of age on this haplotype-dependent regulation of the hAT1R. Aged (>18 months), male mice were used for this part of the study. hAT1R expression increases with age in both haplotypes; however, this increase is significantly higher in Hap I-TG mice (3.17±0.5 to 5.5±0.75 fold) as opposed to mice with Hap II (1.1±0.2 to 1.8±0.1 fold). Age-associated change (Δ) in inflammatory and redox markers was significantly (p<0.05) greater in TG mice with Hap I including, IL1 (4.6±0.8 vs. 2.1±0.49 fold), IL6 (4.0±0.69 vs. 2.1±0.2 fold) and NOX1 (8.3±0.4 vs. 2.5±0.6 fold). This is accompanied by age-associated reduction in levels of antioxidant defenses (SOD1: 0.97±0.0 vs. 1.4±0.1 fold; HO1: 0.77±0.1 vs. 1.3±0.2 fold) and pro-survival genes including, NAMPTS (2.1 folds lower in Hap I vs. Hap II) and SIRT1 (1.8 fold lower in Hap I vs. Hap II). Thus, haplotype-dependent transcriptional regulation of the hAT
1
R gene causes increased hAT1R expression and blood pressure, in Hap I TG mice. Importantly, aging exacerbates this differential gene-expression regulation, further increasing hAT1R and promoting a prooxidant/inflammatory milieu in mice with Hap I.
Abstract 022: Evidence for AT2-receptor-MAS Dimerisation from Fluorescence Resonance Energy Transfer (FRET) Imaging and Fluorescence Cross Correlation Spectroscopy (FCCS)
Authors: Villela Daniel C , Teichmann Anke , Kirsch Sebastian , Mardahl Maibritt , Münter Lisa M , Lucht Kristin , Alenina Natalia , Unger Thomas , Bader Michael , Schülein Raph , Santos Robson A , Steckelings Ulrike M
Published: July 2021

The angiotensin AT2-receptor (AT2R) and the receptor MAS share a strinkingly similar spectrum of signaling mechanisms and protective, physiological actions. Furthermore, cross-inhibition by the respective receptor antagonists has been observed. Therefore we hypothesised that a physical interaction between these two receptors may exist.

HEK-293 cells were transfected with vectors encoding MAS or AT2R fused in the C-terminus with the fluorophores CFP or YFP for FRET and GFP or mCherry for FCCS.

FRET with photobleaching was used to detect, whether MAS and AT2R are localised in very close proximity (1-10nm) in cell membranes thus indicating dimerisation. FCCS was used to follow simultaneously occurring fluctuations in fluorescence intensity of both labeled molecules.

Several controls were applied such as co-transfection of equal amounts of fused and non-fused MAS/AT2R expression vectors for competition, co-tranfection of coding and uncoding pcDNA vectors or co-transfection with an unrelated transmembrane receptor.

Experiments were conducted under baseline conditions and in cells treated with AT2R/MAS agonists and antagonists

Significant FRET efficiency of 10.8±0.8% was measured for AT2-YFP/MAS-CFP strongly indicating heterodimerisation. FRET efficiency was not altered by AT2R or MAS agonists or antagonists. Non-fluorescent MAS and AT2R competed with fluorescent receptors as indicated by a 50% reduction in FRET efficiency (6.0±0.6%), while empty vectors did not compete (9.6±0.6%). No FRET efficiency was observed with an unrelated transmembrane receptor (0.44±1.44%) indicating specificity of receptor interactions. Both, MAS and AT2R also formed homodimers (7.4±0.8% for MAS, 9.2±0.8% for AT2R). Hetero- and homodimerisations were absent if amino acid C35 of the AT2R was mutated (3,9 ± 1,2%).

FCCS corroborated the FRET results and revealed a significantly enhanced cross correlation in cells tranfected with fluorophore-tagged MAS/AT2R when compared to vectors only expressing the fluorophores (8.5±3% vs 11.1±4%; p<0.0001).

Our data strongly suggest that MAS and the AT2R form homo- and heterodimers. Studies to investigate the physiological relevance of MAS/AT2R dimerisation are currently being conducted.
Abstract 042: Effects of Diuretics on Sglt2 Inhibitor-induced Changes in Urinary Sodium Excretion Rate In Obese Metabolic Syndrome Rats
Authors: NISHIYAMA AKIRA , Fujisawa Yoshihide , Sufiun Abu , Rafiq Kazi , Nakano Daisuke , Kobori Hiroyuki , Rahman Asadur
Published: July 2021

Proximal tubular sodium-glucose co-transporter 2 (SGLT2) transports glucose and sodium with a 1:1 stoichiometry. However, studies have indicated that treatment with SGLT2 inhibitors does not substantially increase the urinary excretion rate of sodium, while that of glucose is markedly increased. These data suggest that urinary sodium is reabsorbed by other mechanisms distal to the nephron during SGLT2 inhibition. Here, we aimed to investigate whether diuretics affect the SGLT2 inhibitor-induced changes in the urinary excretion rate of sodium in obese metabolic syndrome SHR/NDmcr-cp(+/+) (SHRcp) rats. Male 13-week-old SHRcp rats were treated with: 1) vehicle (0.5% carboxymethylcellulose), 2) a SGLT2 inhibitor, luseogliflozin (10 mg/kg/day, p.o.), 3) luseogliflozin + hydrochlorothiazide (10 mg/kg/day, p.o.) or 4) luseogliflozin + hydrochlorothiazide + furosemide (5 mg/kg/day, p.o.) for 5 weeks (n = 6-8 per group). Blood pressure and glucose metabolism were evaluated by telemetry and oral glucose tolerance test respectively. Vehicle-treated SHRcp rats developed non-dipper type hypertension (night and day time systolic blood pressure; 186 ± 2 and 185 ± 2 mmHg, respectively) and insulin resistance. As compared with vehicle-treated animals, luseogliflozin-treated rats showed an approximately 4,000-fold increase in urinary glucose excretion and improved glucose metabolism. Luseogliflozin also slightly decreased blood pressure, which was associated with an approximately 30% increase in urinary excretion of sodium. The addition of hydrochlorothiazide or hydrochlorothiazide + furosemide further decreased blood pressure and improved blood pressure circadian rhythm to a dipper profile in luseogliflozin-treated animals. In these animals, urinary sodium excretion tended to be increased by diuretics, although these changes were not statistically significant. These data indicate that SGLT2 inhibitor-induced natriuresis is not enhanced by diuretics. Thus, SGLT2 inhibitors may elicit their beneficial effects on glucose metabolism and hypertension in patients who are treated with diuretics.
Abstract 043: Acute K+ Intake Does Not Reduce Renal Na-Cl Cotransporter Phosphorylation during Chronic AngII Hypertension
Authors: Han Jiyang , Lee Donna , Veiras Luciana , McDonough Alicia A
Published: July 2021

In Sprague Dawley (SD) rats, a single K
+
rich meal provokes a 50% decrease in DCT Na-Cl cotransporter phosphorylation (NCCp) which shifts Na
+
reabsorption downstream for reabsorption by CCD ENaC which drives a homeostatic increase in K
+
secretion. During Ang II dependent hypertension, DCT NCCp increases 2-4 fold as does NCC mediated Na
+
reabsorption. Motivated by these two recent findings, we tested the hypothesis that NCCp elevated by AngII hypertension (400 ng AngII/kg/min x 14 d in SD rats) could be reduced by a single K
+
rich meal (3 hr 2% K meal). Three groups (n=5-6) of male SD rats were compared: 1) Untreated fed 0%K meal, 2) AngII infused fed 0%K meal, and 3) AngII infused fed 2%K meal. By immunoblot of renal cortical homogenates, AngII infusion increased the following 2-5 fold compared to the untreated group: NCC, NCCpS71, NCCpT53, NCCpS89, SPAK, SPAKpS373, and cleaved ENaC-gamma. A single 2%K meal did not reduce abundance or phosphorylation of NCC, SPAK or ENaC cleavage. Interestingly, the AngII infused rats exhibited signs of kaliuresis and K
+
depletion compared to the untreated group: overnight urine K
+
(UKV) was 4.3 +/- 0.1 vs. 2.3 +/- 0.3 mM, fasting plasma [K
+
] was 3.3 +/- 0.1 vs. 3.9 +/- 0.1 mEq in AngII vs. untreated, and while plasma [K
+
] climbed to 4.6 +/- 0.1 after the 2% K meal in AngII infused rats, UKV was not elevated compared to AngII rats fed a 0%K meal. The results indicate that a single K
+
rich meal does not reduce NCCp in AngII infused rats, likely due to our finding that the AngII hypertension protocol provokes kaliuresis and K
+
deficiency. Thus, the ingested K
+
replenishes K
+
stores and is not excreted. The results also suggest that the rise in NCCp during AngII hypertension may be, at least in part, a response aimed to reduce K
+
excretion.
Abstract 049: Role of Smooth Muscle BMAL1 in Time-of-day Vasoconstriction Variation and Blood Pressure Circadian Rhythm
Authors: Xie Zhongwen , Su Wen , Liu Shu , Zhao Guogang , Esser Karyn , Schroder Elizabeth A , Lefta Mellani , Guo Zhenheng , Gong Ming C
Published: July 2021

The blood pressure circadian rhythm was believed to be primarily controlled by the central pacemaker suprachiasmatic nucleus (SCN). This dogma was challenged by the discoveries that each of the clock genes present in the SCN are also expressed and function in peripheral tissues. But whether, and if so how, the peripheral clock genes are involved remains uncertain. The current study investigates the role of Bmal1, an obligatory core clock gene, plays in smooth muscle and blood pressure regulation by using a smooth muscle specific BMAL1 knockout mouse model (SM-Bmal1-KO). The results show:
1)
Smooth muscle specific deletion of BMAL1 does not affect the clock genes in SCN but drastically suppresses the amplitude and the time-of day differences in vasoconstriction in response to various agonist stimulation and to perfusion pressure increase in isolated small mesenteric arteries and pressor responses in anesthetized mice;
2)
The inhibition of agonist-induced vasoconstriction is associated with suppression of MLC
20
phosphorylation, ROCK2 mRNA and activity. Moreover, BMAL1 directly binds to ROCK2 promoter in a time-of-day dependent manner in mesenteric arteries and is required for ROCK2 promoter activity in cultured vascular smooth muscle cells;
3)
Mice lacking smooth muscle BMAL1, but not those lacking cardiomyocyte BMAL1, exhibits alterations in blood pressure. SM-Bmal1-KO mice have moderately but significantly decreased blood pressure under 12:12 light/dark cycle, constant dark, and constant light conditions. The blood pressure circadian rhythm in SM-Bmal1-KO mice has diminished amplitude, forward shifted acrophase, but normal period length and normal locomotor activity;
4)
interestingly, pulse pressure is markedly elevated and the pulse pressure circadian rhythm is abolished in the SM-Bmal1-KO mice. These data provide novel mechanistic insights into the daily control of vasoconstriction and blood pressure, which are fundamentally significant for the elucidation of pathogenesis of diseases involving blood pressure circadian rhythm disruption.
Abstract 055: Locating Genetic Determinants Of Translational Significance Using The Rat Genome
Authors: Nie Ying , Kumaraswamy Sivarajan , Cheng Xi , Waghulde Harshal , Mel Blair , Pillai Resmi , Joe Bina
Published: July 2021

Genome-wide association studies (GWAS) have detected associations of genetic elements on human chromosome 2 with hypertension but lack the ability to discern whether these associations are/are not causal factors for hypertension. Using rat genetic models of hypertension, we have identified that factors linked to the inheritance of hypertension map to rat chromosome 9, which is homologous to human chromosome 2. Here we report that by applying high resolution mapping approaches, we have prioritized two regions on rat chromosome 9, one spanning < 788kb and a second region spanning < 81.8kb, as genomic segments containing novel inherited genetic elements controlling blood pressure. The <788kb region contains 3 protein coding genes,
Trmp8
,
Spp2
and
Arl4c
, which are also associated with human hypertension. Of these, there was only one nonsynonymous polymorphism within the gene
Spp2
between the Dahl S and S.SHR strains used to map this locus. The expression of
Spp2
was significantly higher in the S.SHR rat compared with S. We therefore prioritize
Spp2
as a positional candidate for the <788kb region. The <81.8kb region contains no known/predicted protein-coding genes. We hypothesized that polymorphisms within gene regulatory elements underlie the <81.8kb locus. By combining the locations of the relevant polymorphisms with promoters predicted by the Proscan promoter prediction software, 5 regions were prioritized for further analysis. Alleles from the normotensive R rat from 1 out of these 5 regions had a 5.14 fold higher luciferase activity compared with that of the hypertensive S rat alleles (p<0.05). This region maps from chr9:80880396 to chr9:80882643 (Rnor5.0) on the rat genome. The downstream target for this promoter activity is unknown. Interestingly, within 110,419bp downstream of this region, there is a predicted long non-coding RNA in the mouse (AK079660) and in humans (
DIRC4)
. By RT-PCR using rat kidney, we detected a novel rat transcript that is potentially a rat homologue of the mouse and human predictions. Collectively, these data point to conserved syntenic regions in rats and humans that contain novel promoter sequence variants and variants within the conserved gene,
Spp2
, as potential quantitative trait nucleotides for blood pressure regulation.
Abstract 056: Integrative Genomic Analysis Unravels Novel Pathways And Key Regulatory Networks In Blood Pressure
Authors: Zhao Yuqi , Shi Xingyi , Mäkinen Ville-Petteri , Meng Qingying , Yang Xia
Published: July 2021

Blood pressure (BP) is a highly heritable trait and hypertension is a major risk factor for cardiovascular diseases. Recent genome-wide association studies (GWAS) have implicated a number of susceptibility loci for systolic (SBP) and diastolic (DBP) blood pressure. However, these loci together only explain 1% of the BP variability and the underlying mechanisms remain elusive. In this study, we utilized an integrative genomics approach that leveraged multiple genetic and genomic datasets including 1) GWAS from CHARGE (The Cohorts for Heart and Aging Research in Genomic Epidemiology) Consortium for DBP and SBP, 2) expression quantitative trait loci (eQTLs) from genetics of gene expression studies of human tissues related to hypertension (such as peripheral blood, whole blood, human aortic endothelial cells, and adipose tissue), 3) knowledge-driven biological pathways, and 4) data-driven regulatory gene networks. The integration of these diverse data sources enabled tissue-specific investigations on whether the genetic variants associated with BP concentrated on specific parts of gene regulatory networks, termed as “subnetworks”, and whether novel key regulators in the subnetworks could be identified based on data-driven network topology. We identified 10 and 8 subnetworks for DBP and SBP respectively. Among these, subnetworks associated with ion homeostasis, ALK in cardiac myocytes, B cell receptor signaling, and Regulation of Insulin Secretion, were shared between DBP and SBP. More interestingly, we detected tissue-specific pathways, for example, L1CAM interactions in aortic endothelial cells and Leukocyte transendothelial migration in adipose. Among the trait-specific subnetworks, those involved in megakaryocyte development and cytoskeleton remodeling were found to be SBP-specific while GAB1 signalosome subnetwork was DBP-specific. Finally, by utilizing the gene-gene relationships revealed by the network architecture, we detected key regulator genes, both known (e.g. COL1A1, KL, and OSR1) and novel (e.g. EFEMP1, and CRABP2), in these blood pressure subnetworks. Our results shed lights on the complex mechanisms underlying blood pressure and highlight potential novel targets for hypertension and cardiovascular diseases.
Abstract 057: High Salt (NaCl) Reduces The Activation And Function Of Alternatively Activated (M2) Macrophages Via Epigenetic Modifications
Authors: Binger Katrina , Gebhardt Matthias , Heinig Matthias , Rintisch Carola , Schroeder Agnes , Neuhofer Wolfgang , Hilgers Karl , Manzel Arndt , Schwartz Christian , Kleinewietfeld Markus , Linker Ralf , Lang Florian , Voehringer David , Huebner Norbert , Dechend Ralf , Jantsch Jonathan , Titze Jens , Muller Dominik
Published: July 2021

High intake of dietary salt (sodium chloride; NaCl) is attributed to the development of hypertension and cardiovascular disease, and has been proposed to be a cause for the rapid increase in autoimmune diseases in western civilisations. We have recently shown that NaCl has a pro-inflammatory effect and boosts the activation of Th17 cells in vitro, with mice fed a high salt diet having an accelerated and more severe experimental autoimmune encephalomyelitis. In this study, we examine how the activation of alternatively activated (M2) macrophages is affected by NaCl. In stark contrast to our study with Th17 cells, we find that high salt dose-dependently decreased M2 activation in IL-4+IL-13 stimulated BM-derived mouse macrophages. Genes important for M2 activation, including Mrc1, Arg1, Ym1, Fizz1 and PD-L2, all had a blunted expression in the presence of NaCl; an effect which was not observed in tonicity controls (mannitol or urea), implying a specific action of NaCl. Transcription factors important for modulating M2 function (Irf4 and Klf4) were similarly affected. In contrast to our previous findings in Th17 cells, the effect of salt on M2 activation was not mediated via Sgk-1. To explore the mechanism for the effect of NaCl on M2 activation we performed gene expression analysis simultaneously with genome wide epigenetic modification analysis (ChIP-seq for H3K4me3 and H4ac). The results of this revealed that NaCl modulated epigenetic marks at genes important for M2 activation, and additionally identified new genes which were affected. Finally, we asked if these salt-mediated changes in M2 gene profiles, translated into effects on their function. Using an in vitro assay, we found that NaCl-treated M2 macrophages have a reduced ability to suppress the activation of effector T cells. Our study reveals a novel effect of NaCl on M2 activation and function, and gives support to the notion that the modulation of immune cell function by high dietary salt is relevant to hypertension and autoimmune diseases.
Abstract 058: Sirt3 Impairment and SOD2 Hyperacetylation Drive Vascular Oxidative Stress and Hypertension
Authors: Nazarewicz Rafal , Dikalova Anna , Fessel Joshua P , Itani Hana , McMaster William , Harrison David G , Dikalov Sergey
Published: July 2021

Aging is associated with increased incidence of hypertension and a decline of the mitochondrial energy regulator deacetylase Sirt3. A major mitochondrial antioxidant enzyme, SOD2, is inhibited by acetylation and its deacetylation by Sirt3 restores SOD2 activity. We hypothesized that loss of Sirt3 activity increases vascular oxidative stress due to SOD2 hyperacetylation and that this promotes hypertension. The combination of angiotensin II and TNFα, which is produced by inflammatory cells in hypertension, synergistically reduced Sirt3 expression, induced SOD2 acetylation and increased mitochondrial superoxide (O
2

•

-
) in human aortic endothelial cells (HAEC). mitoEbselen, which scavenges mitochondrial H
2
O
2
, prevented SOD2 acetylation in HAEC and normalized mitochondrial O
2

•

-
suggesting redox-dependent modulation of Sirt3. In intact mice, chronic AngII infusion (490 ng/kg/min) reduced vascular Sirt3 expression by 20%, caused Sirt3 S-glutathionylation, SOD2 hyperacetylation and reduced SOD2 activity by 42%. Mice transgenic for mitochondria-targeted catalase exhibited reduced Sirt3 S-glutathionylation, no SOD2 hyperacetylation, maintenance of SOD2 activity and no increase in mitochondrial O
2

•

-
. The functional role of Sirt3 S-glutathionylation was further supported by mitoEbselen treatment of WT and Sirt3
-/-
mice after the onset of AngII-induced hypertension. mitoEbselen reduced vascular oxidative stress and hypertension in WT but not in Sirt3
-/-
mice. In Sirt3
-/-
mice, low dose AngII (200 ng/kg/min) caused greater hypertension (150 mm Hg) than in WT (128 mm Hg, P<0.01) and AngII-infused Sirt3
-/-
mice produced less nitric oxide that WT, as detected by electron spin resonance (70 pmol/aorta vs 110 WT+AngII, P< 0.01). We further showed that treatment with the mitochondria targeted SOD2 mimetic mitoTEMPO, initiated after the onset of AngII-induced hypertension lowered blood pressure and improved vasodilatation in both WT and Sirt3
-/-
mice, further supporting a role of mitochondrial O
2

•

-
in hypertension. These data indicate that reduced Sirt3 activity occurs in hypertension due to S-glutathionylation and that this leads to SOD2 hyperacetylation and inactivation, promoting vascular oxidative stress and blood pressure elevation.
Abstract 065: Soluble Dipeptidyl Peptidase-4 Induces Endothelial Dysfunction by the Release of Vasoconstrictor Prostanoids: Protective Effect of Dipeptidyl Peptidase Inhibitors
Authors: Peiró Concepción , Romacho Tania , Wronkowitz Nina , Villalobos Laura A , Sell Henrike , Eckel Juergen , Sánchez-Ferrer Carlos F
Published: July 2021

Soluble dipeptidyl peptidase (sDPP)-4 is a novel adipokine whose release is increased in patients with the metabolic syndrome. This study aimed to explore the capacity of sDPP-4 to directly impair vascular reactivity, which is considered as an early hallmark of endothelial dysfunction. As determined using a small vessel myograph, sDPP-4 (20-500 ng/mL) did not affect the contractility to noradrenaline (3 nmol/L to 30 μmol/L) in isolated murine mesenteric microvessels. sDPP-4 did not modify either the endothelium-independent relaxations induced by sodium nitroprusside (1 nmol/L to 100 μmol/L) in microvessels precontracted with 3 μmol/L noradrenaline. However, sDPP-4 impaired in a concentration-dependent manner the endothelium-dependent relaxation elicited by acetylcholine (ACh; 1 nmol/L to 10 μmol/L) with pD2 values of 6.88±0.17, 6.86±0.25, 6.24±0.18, 5.54±0.31 for 0, 20, 100 and 200 ng/mL sDPP-4, respectively. At 500 ng/mL sDPP-4, the maximal relaxation induced by ACh was reduced from 78.20±8.83% to 20.29±3.94%. The inhibition of cyclooxygenase or the blockade of thromboxane TP receptors with indomethacin (10 μmol/L) and SQ29548 (100 nmol/L), respectively, prevented the impaired relaxation to ACh evoked by a submaximal concentration of sDPP-4 (200 ng/mL). In line with these results, sDPP-4 (500 ng/mL) stimulated the release of thromboxane A2 (TXA2) by cultured human coronary artery endothelial cells (HCAEC) from 1.64±0.21 to 3.60±0.70 pg/mL. The DPP-4 inhibitors K579 (100 nmol/L) and linagliptin (10 nmol/L) prevented both TXA2 release and the impaired relaxation caused by sDPP-4. In conclusion, sDPP-4 arises as a causative agent of endothelial dysfunction through the release of COX-derived vasoconstrictor prostanoids. By interfering with such deleterious action of sDPP-4, pharmacological DPP-4 inhibitors might help preventing impaired endothelial function in type 2 diabetes mellitus or the metabolic syndrome.
Abstract 098: Foxp3+ T Regulatory Lymphocytes Counteract Angiotensin Ii-induced Vascular Injury
Authors: Mian Muhammad Oneeb Rehman , Barhoumi Tlili , Briet Marie , Ene Adriana Cristina , Rehman Asia , Paradis Pierre , Schiffrin Ernesto L.
Published: July 2021

Objective:
T effector lymphocytes contribute to vascular injury in angiotensin (Ang) II-induced hypertension, but the role of T regulatory lymphocytes (Tregs) is unclear. Ang II-induced hypertension is blunted in T and B lymphocyte-deficient (Rag1-/-) mice, and restored with reconstitution of T cells. We hypothesized that adoptive transfer of FOXP3-deficient Scurfy (Sf) vs. wild-type (WT) T cells will exacerbate Ang II-induced vascular damage in Rag1-/- mice.

Methods:
Eleven-week old male Rag1-/- mice were injected IV with vehicle, 10 million WT or Sf T cells, 1 million CD4+CD25+ Tregs alone or with Sf T cells, and 2 weeks later were infused or not with Ang II (490 ng/kg/min, SC) for 14 days (n=3-8). Telemetric BP, vascular function and structure, and reactive oxygen species (ROS) production and fibronectin expression in mesenteric arteries (MA) were determined.

Results:
Ang II induced a 40 mmHg systolic BP rise in all the groups, but diastolic BP rise was ~10 mmHg greater in WT and Sf T cell-injected mice than in controls (P<0.01). Treg injection alone or with Sf T cells prevented or delayed by 7 days the BP rise, respectively (P<0.05). Ang II did not induce endothelial dysfunction in vehicle or Treg only-injected mice. Adoptive transfer of WT T cells restored Ang II induced-endothelial dysfunction (60±5% vs. 83±4%, P<0.05), which was exaggerated in Sf T cell-injected mice (56±6% vs. 97±7%, P<0.01), but reduced by Treg co-injection (74±4%, P<0.05). Ang II increased ROS production in MA wall (239±32% vs. 119±20%) and perivascular fat (369±39% vs. 84±8%) in Sf T cell-injected mice (P<0.01), but not when co-injected with Tregs. Ang II induced increased vascular stiffness (P<0.01) and media/lumen (M/L, P<0.05) in vehicle (strain at 140 mmHg: 0.60±0.02 vs. 0.80±0.02; M/L: 4.1±0.2 vs. 2.9±0.2%) and Sf T cell-injected mice (strain at 140 mmHg: 0.63±0.01 vs. 0.89±0.04; M/L: 4.7±0.3 vs. 2.9±0.1%). Ang II increased MA fibronectin expression (P<0.01) in vehicle (113±12 vs. 51±14 RFU/μm2) and Sf T cell-injected mice (85±7 vs. 36±9 RFU/μm2).

Conclusion:
These results demonstrate that Foxp3+ Tregs have a protective role against Ang II-induced vascular dysfunction, remodeling and oxidative stress.
Abstract 210: Impaired Brain-Derived Neurotrophic Factor-TrkB Signaling in Nucleus Tractus Solitarius During Chronic Heart Failure Blunts Baroreflex Sensitivity
Authors: Becker Bryan K , Wang Hanjun , Zucker Irving H
Published: July 2021

Blunted baroreflex function is a negatively prognostic marker of autonomic imbalance in chronic heart failure (CHF), yet the mechanisms underlying baroreflex blunting in CHF are not fully understood. The nucleus tractus solitarius (NTS) is the primary central target of baroreceptor afferents, and plays a critical role in regulating central baroreflex sensitivity. Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are highly expressed in the NTS, but little is known about the role of BDNF signaling in the NTS. We hypothesized that in the NTS, BDNF enhances baroreflex sensitivity, and impaired BDNF-TrkB signaling contributes to the blunted baroreflex function in CHF. To test this hypothesis, 50 nl of BDNF or the selective TrkB antagonist ANA-12 were microinjected bilaterally into the dorso-medial NTS of sham-operated rats and myocardial infarct-induced CHF rats. BDNF evoked a greater decrease in blood pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) in sham vs. CHF rats. Injection of ANA-12 increased MAP, HR, and RSNA to a greater extent in sham vs. CHF rats (Table). Following 125 μM ANA-12, baroreflex sensitivity was blunted in sham rats (3.7 ± 0.3 vs. 1.9 ± 0.1 bpm/mmHg max gain, p < .05 before vs. after, n = 4). CHF rats had blunted baroreflex sensitivity vs. sham (2.3 ± 0.1 bpm/mmHg, p < .05, n = 4), and ANA-12 had little effect on baroreflex sensitivity in CHF (2.0 ± 0.1 bpm/mmHg, n = 4). These data suggest that endogenous BDNF plays an important role in maintaining baroreflex sensitivity in the NTS, and that BDNF-TrkB signaling is impaired in CHF, which may contribute to blunted baroreflex sensitivity and autonomic imbalance.
Abstract 213: Fat Tissue specific Adipose Triglyceride Lipase as a major determinant for the development of pressure overload-induced heart failure
Authors: Salatzki Janek , Brix Sarah , Ban Zsofia , Fliegner Daniela , Benz Verena , Kershaw Erin E , Kintscher Ulrich , Foryst-Ludwig Anna
Published: July 2021

Introduction:
Myocardial metabolism undergoes change in response to pathological cardiac hypertrophy (PH), characterized by increased reliance on glucose oxidation, decreased free fatty acid (FFA) oxidation and a loss of metabolic flexibility. Cardiac metabolism is influenced by other organs such as adipose tissue. Hence, we aimed to investigate the effect of Adipose Triglyceride Lipase (ATGL) in adipose tissue on the development of PH and heart failure (HF) in a pressure overload-induced cardiac hypertrophy model in mice.

Methods:
Male adipose tissue specific ATGL-knock out (atATGL-KO) and wild type mice (WT) underwent sham surgery (sham) or transverse aortic constriction (TAC). After 11 weeks, mice were sacrificed and organs were harvested.

We performed echocardiography one week before and 11 weeks after surgery. Left ventricular mass (LVM), left ventricular mass/tibia length (LVM/TL) and ejection fraction (EF) were calculated. Beta-myosin heavy chain (β-MyHC) was measured in RNA of hearts. Insulin resistance was assessed by an intraperitoneal glucose tolerance test (GTT) and an insulin tolerance test (ITT). FFAs were measured in serum in total.

Results:
LVM and LVM/TL in WT was significantly higher compared to atATGL-KO after TAC (LVM/TL [mg/mm] WT-TAC: 18,0±2,2; atATGL-KO-TAC: 13,1±2,3; p<0,01). The higher increase of LVM in WT was associated with a larger left ventricle internal diameter. Reduction of EF was significantly more pronounced in WT compared to atATGL-KO ([%] WT: 28,81±6,9 atATGL-KO: 42,39±4,5; p<0,01).

Beta-MyHC, a marker for PH, was markedly higher in WT-TAC than in atATGL-KO-TAC (WT-TAC: 11,3±3,6; atATGL-KO-TAC: 1.9±0,6; p<0,01).

While WT-TAC showed higher Serum FFA-levels than atATGL-KO-TAC ([mmol/l] WT-TAC: 0,97±0,086; atATGL-KO-TAC: 0,49±0,032; p<0,001), GTT and ITT revealed a higher insulin sensitivity in atATGL-KO-TAC compared to WT-TAC.

Conclusion:
The present study demonstrates that atATGL is a crucial determinant for the development of pressure overload-induced PH and HI. The lack of ATGL in adipose tissue, the associated reduction of fatty acid release in the circulation and subsequent switches in cardiac energy substrates from free fatty acids to glucose are potential underlying mechanisms of this process.
Abstract 233: Angiotensin-(1-7) Abolishes AGE-Induced Chronic ERK Activation and Myofibroblast Transition
Authors: Alzayadneh Ebaa M , Chappell Mark C
Published: July 2021

Advanced glycated end products (AGEs) including glucose and methylglyoxal-modified albumin (MGA) may play a key role in tissue injury arising from various pathologies. Activation of AGE receptor (RAGE) induces myofibroblast transition (MT), hypertrophy and tubulointerstial fibrosis associated with diabetic nephropathy. AGE-RAGE signaling evokes activation of the renal angiotensin system (RAS) and the Ang II-AT1 receptor may facilitate the actions of AGE; however, the role of the Ang-(1-7)-Mas receptor axis in the AGE pathway is unknown. Moreover, there are reports that Ang-(1-7) directly induces renal MT and fibrosis. Therefore, the current study addressed the functional influence of Ang-(1-7) in AGE-induced signaling in the NRK-52E proximal tubule cell line following MGA treatment for 48-72 hours. MGA exposure enhanced cellular hypertrophy 175% (p<0.05 vs. control), increased α-SMA protein 300% (0.4±0.1 vs. 0.1±0.1U; p<0.05), stimulated myofibroblast transition (MT) and markedly increased the cellular release of TGF-β 350% (1.2±0.1 vs. 0.3±0.1 ng/ml; p<0.05). Ang-(1-7) (100 nM) essentially abolished the MGA-induced cell hypertrophy (175±0.1% vs. 85±0.1%), α-SMA expression (0.4±0.1 vs. 0.1±0.1U; p<0.05), and MT, but did not attenuate the increase in TGF-β release (1.2±0.1 vs. 1.3±0.1 ng/ml). Since TGF-β and MAP kinases are integral to the AGE-RAGE signaling cascade, we assessed the effect of TGF-β receptor kinase (SB525334, SB) and ERK1/2 (PD98059, PD) inhibitors. Similar to Ang-(1-7) treatment, SB and PD abolished MGA-induced hypertrophy, α-SMA expression and MT. We further show that both Ang-(1-7) and SB abolished the chronic activation of ERK1/2 at 48 hour MGA exposure. Finally, Ang-(1-7) abolished the TGF-β stimulation of ERK1/2. Pre-treatment of the MGA exposed cells with the AT7/Mas receptor antagonist D-Ala7-Ang-(1-7) reversed the inhibitory actions of Ang-(1-7), but did not potentiate the responses. In summary, we report that Ang-(1-7) abolished AGE activation of TGF-β and ERK1/2 pathways in the NRK-52E cells. We conclude that Ang-(1-7) may provide a potential therapeutic approach in addition to current RAS blockade regimens to prevent the progression of diabetic injury and the decline in renal function.
Abstract 242: Prevalence Of Hypertension And Associated Anthropometric Risk Factors In Indigenous Adults Of Guatemala
Authors: ORELLANA-BARRIOS MENFIL A , Nugent Kenneth , Pena Camilo , Sanchez-Barrios Herman , Lopez-Gutierrez Jose R
Published: July 2021

Background:
Hypertension epidemiological studies in developing regions of the world like rural Guatemala are lacking.

Methods:
A sample size of 1,104 subjects (552 females, all 18 years or older) was obtained through quota and geographical clustering in the entire Department of Sololá, Guatemala. Descriptive statistics and logistic regression were used.

Results:
Average systolic, diastolic and mean arterial pressures were significantly higher in men compared to women (116.24 vs. 113.80 mmHg, 75.24 mmHg vs. 72.69 mmHg, and 88.91 mmHg vs. 86.39 mmHg, respectively; all with p<0.05). The crude prevalence of HT was 12.5 % with no gender differences. Women had a significantly higher mean BMI than men (26.25 vs. 24.71, p< 0.001). An abnormally high WC was found in 12.7% of men and in 50.7% of women. Significant associations were found between the presence of HT, age ≥ 55 years and an elevated WC. The single most important isolated risk factor for HT was age in women (OR 6.76, CI 95% 3.59-12.72) and WC in men (OR 3.23, CI 95% 1.52-6.87). Increased BMIs (≥ 25 to 30 or ≥30) were not associated with HT in this study. Residing in Sololá's capital was a protective factor in women (OR 0.33, CI 95% 0.13-0.83).

Conclusion:
Hypertension and associated anthropometric risk factors are present in rural regions of Guatemala. Significant associations are found between gender, age ≥ 55 years, and increased WC but not with an increased BMI in this population.