bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 91: Activation of The Antioxidant Response Element (ARE) in Human Renal Glomerular Endothelial Cells (HRGECs) Reduces Asymmetric Dimethylarginine (ADMA) And Increases Nitric Oxide by Transcriptional Activation of Dimethylarginine Dimethylaminohydrolase (DDAH) and Endothelial Nitric Oxide Synthase (eNOS)
Authors: luo Zaiming , Aslam Shakil , Bhupatkar Jenny , Welch William J , Wilcox Christopher S
Published: July 2021

The E2-related factor 2 (Nrf2) binds to AREs and transcriptionally activates several genes that protect against oxidative stress. DDAH phosphorylates and activates eNOS and degrades ADMA which is an endogenous inhibitor of eNOS. We detected 2-3 putative AREs in the promoter regions of the human genes for DDAH-1 and -2, peroxisome proliferator-activated receptor-γ (PPAR-γ) and eNOS. PPAR-γ can increase eNOS expression and eNOS phosphorylation. Therefore, we hypothesized that AREs regulate the DDAH/eNOS/PPAR-γ/ADMA/ NO pathway. Tert-butylhydroquinone (tBHQ) activates Nrf2 by translocation to the nucleus. Incubation of HRGECs for 24 hours with tBHQ enhanced cell proliferation dose-dependently (5-20μM). tBHQ (20μM; n=4) increased medium nitrite by 69 ± 3.7% (P<0.01), intracellular NO (from DAF-FM fluorescence ) by 62 ± 17.1% (P<0.05), and NOS and DDAH activities( from conversion of [

3

H]-arginine or [

14

C]-ADMA to [

3

H]- or [

14
C]

-citrulline) by 58 ± 3.6% and 47 ± 8.5% (P<0.05) and decreased ADMA (by capillary zone electrophoresis) from 1.2±0.03 vs. 2.3±0.16μM/L, (P<0.001, n=6). tBHQ increased the mRNAs and proteins for eNOS by 79 ± 9% and 43 ± 17%, for DDAH-1 by 129 ± 10% and 62 ± 21%, and for DDAH-2 by 119 ± 25% and 48 ± 18% (all P<0.05) and increased the protein expression of PPAR-γ and eNOS phosphorylation and increased tBHQ nuclear translocation markedly. Knockdown of the cell Nrf2 by transfection with a specific siRNA abolished all these effects and attenuated cell proliferation. Chromatin immunoprecipitation (CHIP)-based PCR assays demonstrated that tBHQ enhanced the binding of Nrf2 to one ARE region in the promoters for DDAH-1 and -2 but there was no binding to the eNOS promoter. In conclusion, activation of a specific ARE in the promoter region of the DDAH-1 and -2 genes causes their transcriptional activation. The ensuing enhancement of DDAH activity enhances ADMA metabolism. Nrf2 also increases eNOS expression, NOS activity and NO generation without binding to the ARE for eNOS, likely by upregulation of PPAR-γ. Thus, Nrf2 improves endothelial cell function by coordinating a diverse set of pathways that enhance NO generation.
Abstract 16: Physiological Characterization of a Selective Knockout of the Intracellular Isoform of Renin in the Brain of Mice
Authors: Folchert Matthew D , Littlejohn Nicole K , Liu Xuebo , Grobe Justin L , Sigmund Curt D
Published: July 2021

Renin gene expression is regulated by two distinct promoter-first exon combinations that target renin for either secretion (exon 1a initiating secreted renin, sREN) or for cytoplasmic retention (exon 1b initiating intracellular renin, icREN). The icREN isoform is expressed predominantly in the brain and its expression is downregulated whereas sREN expression is upregulated by deoxycorticosterone (DOCA)-salt suggesting each isoform may be differentially regulated. We generated mice that conditionally lack icREN, but preserve sREN, by targeting a novel renin allele in ES cells by flanking exon-1b and its surrounding sequences (including the promoter) with loxP sites. Mice carrying this allele were first bred with flippase mice to remove the neo cassette and then with cre-recombinase mice to generate the null allele lacking icREN. The structure of the final allele was confirmed by Southern blot. There was no apparent lethality in the homozygous mice when heterozygous mice were intercrossed. Real time quantitative RT-PCR analysis revealed a loss of icREN mRNA in the brain, but a preservation of sREN mRNA in the kidney. Male icREN-KO mice were similar in size (n=4, 14.7±0.9 wk, 27.94±1.08 g) compared to littermate controls (n=7, 14.2±0.8 wk, 27.96±0.82 g, P=0.988), but by NMR exhibited reduced total and relative total fat mass (1.83±0.15 vs 3.00±0.12 g, or 6.6±0.7 vs 10.9±0.5 %, both P<0.001). Interestingly, interscapular brown adipose (289±77, n=5 vs 68±61 mg, n=9 P=0.04) and heart (162±12 vs 129±9 mg, P=0.04) masses were both increased in icREN-KO mice, possibly suggesting increased resting metabolic rate (RMR) and blood pressure. Liver and kidney masses were normal. Food intake was normal (3.25±0.28, n=4 vs 3.14±0.14, n=8 P=0.7), but preliminary tests uncovered trends toward increased RMR (7.67±0.75, n=4 vs 6.93±0.59 g/day, n=4 kcal/kg lean/hr, P=0.47) and glucose tolerance (2g/kg, 25768±3994, n=3 vs 33734±2854 mg/dL*min, n=8, P=0.157). Studies are ongoing to assess arterial pressure. Together these data suggest that this novel icREN isoform contributes to metabolic and possibly to cardiovascular control. Future studies using the conditional allele (icREN-flox) will provide an opportunity to dissect the neural circuits involved in these responses.
Abstract 174: Angiotensin Production Specifically Within the Subfornical Organ Induces Polydipsia
Authors: Coble Jeffrey P , Cassell Martin D , Grobe Justin L , Sigmund Curt D
Published: July 2021

Angiotensin (ANG) production within the subfornical organ (SFO) is necessary for polydipsia resulting from elevated RAS activity, yet whether SFO ANG is sufficient to increase fluid intake is unknown. We examined the sufficiency of SFO-ANG to cause polydipsia in a novel double transgenic model. The model consists of 1) synapsin-human renin (sR) specifically expressing human renin in neurons via the synapsin promoter, and 2) a construct expressing dsRED at baseline switched to human angiotensinogen (hAGT) in response to Cre-recombinase. Region-specific expression of ANG in the brain of sRA-Red mice can be induced by injection of an adenovirus encoding Cre-recombinase (AdCre). Inducing expression of hAGT in the SFO by ICV injection of AdCre into sRA-Red mice resulted in a progressive increase in fluid intake reaching a maximum 25-days after injection (sRA-Red: 0.26±0.02; NT: 0.17±0.02 mL/g/day, P<0.001). The increase in total fluid intake of sRA-Red mice elicited by ICV AdCre administration was attenuated by an acute ICV injection of losartan (200 mg: 0.14±0.03 mL/g/day, P<0.001) to a level similar to littermate controls receiving losartan (0.17±0.02 mL/g/day). Polydipsia and hypertension due to elevated brain ANG is known to involve the MAPK pathway through activation of angiotensin type 1 receptor (AT1R). The SFO has anatomically and functionally distinct regions. The core region has been implicated in mediating hypertension, whereas the peripheral region controls drinking behavior. ICV-AdCre-sRA-Red mice exhibited increased numbers of phospho-ERK1/2 (62±15 vs 34±13, P=0.05) and phospho-CREB (85±10 vs 55±8, P<0.05) immuno-positive cells per section in SFO. ERK1/2 phosphorylation showed trends toward increased cells per section within both the periphery (26±7 vs 15±6, P=0.10) and core (21±5 vs 11±4, P=0.05) of the SFO. CREB phosphorylation significantly increased in both the periphery (44±9 vs 21±2, P<0.05) and core (27.4±1.6 vs 21.0±1.8 vs, P<0.05) of the SFO. We conclude that selective production of ANG within the SFO is sufficient to increase fluid intake through central activation of AT1R. We are currently examining arterial pressure and testing if the drinking response occurs through ERK1/2- and CREB-dependent pathways in the SFO.
Abstract 196: Role of Collecting Duct Renin in Blood Pressure Regulation
Authors: Ramkumar Nirupama , Stuart Deborah , Rees Sara , Sigmund Curt , Kohan Donald E
Published: July 2021

Recent studies propose that collecting duct (CD) renin is an important modulator of blood pressure regulation, especially in conditions such as angiotensin-II infused hypertension. We used gene targeting to generate a CD-specific renin knockout (KO) to assess if CD derived renin can regulate BP. Utilizing the Cre lox P system, exon 1 of the renin gene was ablated specifically in the CD. BP was recorded via telemetry and plasma and urine were collected in metabolic cages on normal, high and low Na diets. DNA recombination showed kidney specific recombination in KO mice. Compared to floxed mice, CD renin KO mice had 70 % lower medullary renin mRNA levels and 90% lower renin mRNA in micro-dissected cortical and inner medullary CD tubules. Urinary renin levels were significantly lower in the KO mice on normal and low Na diets (45% of floxed levels) but not with high Na intake. Plasma renin concentration was significantly higher in the KO mice on all three diets. While BP was similar between the two groups on all three diets, infusion of Ang-II delayed the increase in BP in the CD renin KO group for at least 4 days post-infusion. These findings suggest that CD renin likely plays a role in normal BP regulation (evidenced by an increase in PRC) and in response to AngII infusion.
Abstract 199: Estrogen Administration Reduces Blood Pressure in Female, but not in Male Obese Mice
Authors: Wang Yu
Published: July 2021

Objective:
We recently demonstrated that male, but not female mice fed a high fat (HF) diet developed obesity-hypertension. Castration of female mice conferred obesity-hypertension. The hypothesis of this study is estrogen administration reduces obesity-hypertension in intact male and ovariectomized female HF-fed mice.

Methods and Results:
Male (8 weeks old) and ovariectomized female (10 weeks old) C57BL/6 mice were fed a HF diet (60% kcal as fat) for 16 weeks. Exogenous estrogen (36g/ml 17-estradiol) or vehicle (sesame oil) were continuously delivered via silastic tubes implanted subcutaneously in HF-fed male mice from the beginning of HF feeding. For females, HF diet and estrogen administration began 2 weeks after ovariectomy. There was no effect of estrogen administration on the development of obesity in male mice. However, female mice administered estrogen showed a marked reduction in the development of obesity (Body weight: Vehicle: 48.7 ± 1.7 g; Estrogen: 38.9 ± 1.9 g; p<0.05). Administration of estrogen to male HF-fed mice improved glucose tolerance (AUC; estrogen: 42150 ± 1865; Vehicle: 48463 ± 1899; p =0.025). Systolic blood pressure (SBP) was not influenced by estrogen administration in male mice (SBP: Vehicle: 147 ± 4 mmHg; Estrogen: 147 ± 3 mmHg). However, estrogen decreased SBP in female HF-fed ovariectomized mice (SBP: Vehicle: 153 ± 11 mmHg; Estrogen: 137 ± 5 mmHg; p=0.019).

Conclusion:
Administration of estrogen to male obese mice had no effect on blood pressure, but improved glucose homeostasis. In ovariectomized HF-fed females, estrogen administration reduced blood pressure. These results suggest that resistance to obesity-hypertension in females may be estrogen-mediated.
Abstract 227: A Liquid Chromatography-Mass Spectrometry Assay for the Semi-quantitative Screening of 34 Cardiovascular Medications in Human Serum
Authors: McNaughton Candace D , Dias Eduardo , Hachey Brian , Yu Chang , Nian Hui , Straka Britt , Caprioli Richard M , Brown Nancy J
Published: July 2021

Background
Cardiovascular medications are the most commonly prescribed drugs; there is need for an objective method to distinguish patients with ineffective regimens from those with medication non-adherence.

Methods
We developed a liquid chromatography mass spectrometry (LC-MS) method for semi-quantitative drug detection of 34 commonly prescribed cardiovascular drugs using 100μl of serum. Drug classes include: anticoagulants, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, beta blockers, calcium channel blockers, diuretics, statins, vasodilators, digoxin, fenofibrate, and niacin. Analytical accuracy and precision were evaluated by repeating the assay on spiked samples at low, medium and high concentrations. In 294 clinical samples collected from hospitalized patients, statistical sensitivity, specificity, and accuracy were computed by comparing the detection or lack thereof against documented drug administration.

Results
For the 34 cardiovascular drugs, the assay was sensitive (>0.90) for all except captopril (0.25), isosorbide (0.81), and niacin (0.89). The assay was specific for all drugs, with a minimum specificity of 0.94 (aspirin).

Conclusions
To our knowledge, this method is the first high-throughput,semi-quantitative LC-MS method for simultaneous analysis of 34 commonly prescribed cardiovascular drugs across multiple classes that has been validated in serum obtained from patients during supervised medication administration. This assay provides a simple tool to determine adherence.
Abstract 229: Increased Ace Gene Dosage Reduces Ace2 Activity in Diabetic Mice Kidney: Involvement of Ace/ace2 Balance on the Development of Diabetic Nephropathy
Authors: Bertoncello Nadia S , Moreira Roseli P , Yokota Rodrigo , Rosa Rodolfo M , Arita Danielle Y , Aragão Danielle S , Cunha Tatiana S , Casarini Dulce E
Published: July 2021

The mechanisms underlying the link between high constitutive levels of ACE and diabetic nephropathy has not been completely understood, but an imbalance between angiotensin I (ACE) and II (ACE2) converting enzymes homeostasis has been described in diabetic kidney disease. The aim of this study was to evaluate ACE/ACE2 homeostasis in kidney from diabetic mice presenting increased dosage of ACE gene. Male mice (3 months old) genetically engineered to harbor one or three copies of the
ACE
gene were made diabetic (streptozotocin - STZ, 50 mg/Kg) and randomly assigned into: 1-copy control (1CC), 1-copy diabetic (1CD), 3-copy control (3CC) and 3-copy diabetic. At the end of experimental period body weight was evaluated and kidney was excised. Kidney-to-body weight ratio and ACE and ACE 2 activities were determined using specific substrates (ZPhe-HL and 7-Mca-APK(Dnp), respectively) (Two way ANOVA + Tukey test; P<0.05). Diabetes increased blood glucose (1CD : 436 ± 25
vs.
1CC: 90 ± 2; 3CD: 556 ± 6
vs.
3CC: 112 ± 4 mg/dL) and kidney-to-body weight ratio (1CD: 7.5 ± 0.2
vs.
1CC: 5.8 ± 0.2; 3CD: 7.8 ± 0.1
vs.
3CC: 5.8 ± 0.1 mg/g) with no influence of
ACE
genotype. As expected, renal ACE activity was directly related to
ACE
gene copy number in control group (3CC: 9.4 ± 2.11
vs.
1CC:5.6 ± 0.9 mU/mg protein). Renal ACE activity was decreased in diabetic groups (1CD: 3.6 ± 0.2
vs.
1CC: 5.6 ± 0.9; 3CD: 2.3 ± 0.4
vs.
3CC: 9.4 ± 2.1 mU/mg protein) with no influence of
ACE
genotype. Under physiological condition, renal ACE2 activity remained unchanged regardless of the
ACE
genotype (1CC: 1.9 ± 0.2 = 3CC: 1.4 ± 0.1 μM/min/mg). However upon a pathological stimulus, renal ACE2 activity was efficiently increased only in 1CD group, but not in 3CD, as compared with the others (1CD: 5.1 ± 0.9 vs. 1CC: 1.9 ± 0.2 = 3CC: 1.4 ± 0.1 = 3CD: 2.2 ± 0.2 μM/min/mg). Taken together, our results show for the first time, that susceptibility for the development of diabetic nephropathy associated with increased ACE gene dosage may be, at least in part, caused by a decrease on renal ACE2 activity. This may result in increased local levels of angiotensin II and decreased angiotensin (1-7), leading to altered glomerular permeability and albuminuria, functional alterations presented by 3CD animals. Financial Support: FAPESP, CAPES, CNPq.
Abstract 262: Cytochrome P450 1B1 is Critical for the Development of Atherosclerosis and Hypertension in ApoE Knockout Mice
Authors: Ghafoor Khuzema , Ghafoor Hafiz Usama , Jennings Brett L , Khan Nayaab S , Thirunavukkarasu Shyamala , Song Chi Young , Zaidi Sahar , Estes Anne M , Malik Kafait U
Published: July 2021

We have reported that cytochrome P450 (CYP) 1B1 contributes to hypertension in various experimental models and promotes neointimal growth in injured carotid arteries. This study was conducted to determine the role of CYP1B1 in the development of atherosclerosis and its pathogenesis, including reactive oxygen species (ROS) production, inflammation, and hypertension caused by atherogenic diet (AD; TD02028). Eight weeks old male ApoE knockout (

ApoE
-/-
/Cyp1b1
+/+

) and ApoE /CYP1B1 knockout (

ApoE
-/-
/Cyp1b1
-/-

) mice were fed a normal diet (ND) or AD for 12 weeks. A separate group of

ApoE
-/-
/Cyp1b1
+/+

mice on AD, were injected twice weekly with the selective inhibitor of CYP1B1, 2,3',4,5'-tetramethoxystilbene (TMS) (300 μg/kg), or its vehicle DMSO (i.p.). Systolic blood pressure (SBP) was measured weekly by tail cuff. After 12 weeks, animals were sacrificed; blood was collected for lipid analysis and aorta for measurement of lesions by Oil red O, collagen deposition by Masson’s trichrome and ROS production by dihydroethidium staining, and infiltration of macrophages (F4/80) and T-lymphocytes (CD3) by immunohistochemistry. Plasma lipid levels, area of lipids and collagen deposition, ROS production (Table), infiltration of macrophages, and T lymphocytes in the aorta and SBP were increased in

ApoE
-/-
/Cyp1b1
+/+

mice on AD vs. ND; these were minimized in mice on AD given TMS but not its vehicle, and in

ApoE
-/-
/Cyp1b1
-/-

mice on AD. These data suggest that the development of hypercholesterolemia by AD and associated pathogenesis including increase in SBP in

ApoE
-/-
/Cyp1b1
+/+

mice depend on CYP1B1 and that it could serve as a novel target for treatment of atherosclerosis.
Abstract 268: Toll-like Receptor 9 (TLR9) Plays a Key Role in the Autonomic Cardiac and Baroreflex Control of Arterial Pressure
Authors: Rodrigues Fernanda L , Bomfim Gisele F , Silva Carlos A , Fazan Rubens , Tostes Rita C , Carneiro Fernando S
Published: July 2021

Introduction:
The crosstalk between the immune and nervous system can impact cardiovascular regulation. Toll-like receptor 9 (TLR9) is expressed in immune cells, peripheral nerves and vascular smooth muscle cells. TLR9 is involved in hypertension and cholinergic stimulation suppresses immune responses mediated by TLR9. Based on that, we hypothesized that TLR9 plays a role in cardiac autonomic and baroreflex control of arterial pressure (AP).

Methods and Results:
TLR9 knockout (KO) and WT mice were anesthetized with isoflurane, implanted with catheters into left carotid artery and right jugular vein and allowed to recovery for 3 days. After basal recording of AP, mice received cardiac autonomic receptor blockers methyl atropine or propranolol. AP and pulse interval (PI) variability were evaluated using the symbolic analysis (non-linear method). Spontaneous baroreflex was evaluated by sequence technique. Mean AP was slightly higher in TLR9 KO (TLR9 KO: 126±2.8 vs WT: 115±4.4 mmHg). In the symbolic analysis the 0V pattern was lower (TLR9 KO: 20±5.4 vs WT: 35±5.0 %) and 2LV pattern was higher in the TLR9 KO (TLR9 KO: 6±1.6 vs WT: 2±0.5 %), an indication of sympathetic and parasympathetic activity, respectively. AP/PI sequences were similar between groups. However, the gain of AP/PI sequences was increased in TLR9 KO (TLR9 KO: 5±1.4 vs WT: 2±0.4 ms/mmHg). Atropine-induced tachycardia was increased in TLR9 KO (TLR9 KO: 23±8.0 vs WT: 2±0.7 ms), whereas propranolol-induced bradycardia was decreased (28±11.0 vs WT: 56±7.6 ms).

Conclusions:
Our findings demonstrate that TLR9 negatively modulates cardiac vagal tone, and consequently the baroreflex control of AP.
Abstract 273: Increased Blood Pressure and Proteinuria and Absence of Increased Nitric Oxide (NO) Production During Pregnancy in the Dahl Salt Sensitive (S) Rat
Authors: Gillis Ellen E , Mooney Jennifer N , Williams Jan M , Garrett Michael R , Sasser Jennifer M
Published: July 2021

In normal pregnancy, systemic vasodilation due to increased NO production allows a drop in blood pressure (BP) despite increased volume retention. Little is known about the pathogenesis of preeclampsia, defined by increased BP and proteinuria, due to a lack of animal models that spontaneously develop the disease. Here we tested the hypothesis that the Dahl S rat, a genetic model of hypertension and kidney disease, is also a spontaneous model of preeclampsia. Female Dahl S rats were implanted with a telemetry unit, and baseline BP was recorded. Rats were placed in metabolic cages for 24 hr urine collection while on a low nitrate diet, and urinary protein and NO metabolite concentrations were measured via Bradford and Greiss assays, respectively. There were no differences in baseline BP (152±1 vs 151±4 mmHg) or proteinuria (61±10 vs 60±17 mg/d) in the rats selected for mating vs virgin rats (n=5-7). Pregnancy was confirmed by presence of sperm (day 1). Measurements were made during mid and late pregnancy (days 10-11, 17-18), and terminal measurements were taken on day 19. Pregnant rats exhibited an increase in BP and proteinuria with no change in urinary NOx excretion (Table), while no changes were observed in age-matched virgin rats. Kidney cortex abundance of neither NOS1 nor NOS3 was increased at late pregnancy; however, plasma concentration of the endogenous NOS inhibitor ADMA was increased in late pregnant compared to virgin rats (0.82±0.06 vs. 0.62±0.06 μM, p<0.05). These data suggest that the Dahl S rat cannot upregulate NO production during pregnancy; therefore, this relative NO deficiency may contribute to worsening hypertension and proteinuria during pregnancy in this strain.
Abstract 274: Detrimental Role of 5’-ecto-nucleotidase (cd73) in the Pathogenesis of Preeclampsia by Producing Excessive Adenosine
Authors: Iriyama Takayuki , Parchim Nicholas F , Liu Chen , Wang Wei , Blackwell Sean , Kellems Rodney E , Xia Yang
Published: July 2021

Background:
Preeclampsia (PE), a life-threatening hypertensive pregnancy disorder, has long been speculated to be associated with placental hypoxia. Adenosine (Ado) is a signaling molecule well known to be induced by hypoxia. Ado levels are increased in the fetomaternal circulation of PE patients and correlated to its severity. However, the causative factors for its induction and pathophysiologic role of elevated Ado in PE are undetermined.

Methods & Results:
Using RT-PCR profiling of purinergic components in the human placentas, we found that CD73, a key enzyme producing extracellular Ado, was elevated most in the placentas of PE patients. To determine the exact role of elevated CD73 in Ado production, we took advantage of a well-established PE model: pregnant mice infused with angiotensin type 1 receptor agonistic antibody (AT1-AA), an emerging pathogenic factor known to contribute to PE. Similar to human studies, we found that both CD73 expression and Ado levels were significantly increased in the placentas of AT1-AA-injected pregnant mice compared to the controls. To determine whether elevated placental Ado is dependent on increased placental CD73 and contributes to pathophysiology of PE, we took both genetic and pharmacological approaches. We found that AT1-AA-mediated placental Ado production was reduced in CD73-deficient pregnant mice. As such, AT1-AA-induced features of PE including hypertension and proteinuria also significantly reduced (BP: 166 to 144 mmHg, proteinuria: 42 to 31 mg/g creatinine). Similarly, we found polyethylene glycol-linked adenosine deaminase (PEG-ADA), a drug that has successfully treated ADA-deficient patients by lowering Ado, ameliorated PE features significantly (BP: 170 to 141 mmHg, proteinuria: 39 to 28 mg/g creatinine). Mechanistically, we further revealed that HIF-1a is a key transcription factor underlying increased CD73 expression, Ado elevation, and subsequent sFlt-1 induction and disease development.

Conclusions:
Our studies revealed i) a pathologic nature of chronic elevated placental Ado in PE; ii) HIF-1a-dependent CD73 upregulation in placenta underlying Ado induction, sFlt-1 elevation and PE features; and iii) novel diagnostic markers and therapeutic possibilities for the disease.
Abstract 304: The Role of Calcitonin Gene Related Peptide (CGRP) in Cardiovascular Regulation and Blood Vessel Structure
Authors: Mai Tu , Diedrich Andre' , Gangula Pandu , Garland Emily , Robertson David
Published: July 2021

Calcitonin gene related peptide (CGRP) is known to be a potent microvascular dilator and hypotensive agent. This peptide and its receptors are distributed widely in both central and peripheral nervous systems. CGRP-containing neural fibers are closely associated with blood vessels in the arterial circulation where its release leads to vascular dilation. However, its role in cardiovascular regulation remains controversial. We studied the hemodynamic and autonomic function, as well as blood vessel structure in CGRP knockout (KO) mice. Blood pressure (BP), heart rate (HR) and activity level were assessed at least one week after mice were implanted with telemeters. Ten CGRP KO and ten wild type (WT) mice (4-5 month old) were used in this study. After the recording period, 24-hr urine and blood were collected for assessment of catecholamines and their metabolites. To study the baroreflex sensitivity, phenylephrine and sodium nitroprusside were administered in an acute study with seven mice from each group. We found no significant difference in 24-hr mean arterial pressure (MAP) (105.0 ± 1.8mmHg, vs. 112.1 ± 3.1mmHg, p= 0.05) between the two groups. There was also no difference in HR (KO: 566.4 ± 12.1; WT: 581.9 ± 8.5, p = 0.31). Norepinephrine was greatly elevated in both plasma and 24-hr urine in KO mice (in urine: KO: 956 ± 91 pg/ml, WT: 618 ± 46pg/ml, p = 0.004; in plasma: KO: 2505 ± 596pg/ml, n=6, WT: 1168 ± 98 pg/ml, p = 0.04). Paradoxically, cardiovagal baroreflex sensitivity was higher in the KO mice (KO: 3.2 ± 0.7 ms/mmHg, n=7, WT: 1.4 ± 0.3 ms/mmHg, n=7, p=0.03). Thoracic aortas were collected from both groups to measure vessel stiffness. The diameter at 125 mmHg of the aorta was significantly smaller in CGRP KO mice than in WT (321±30μm, vs. 422±82μm, p<0.01). Therefore, we hypothesize that although CGRP may have a limited contribution to physiological cardiovascular regulation, it may play a significant role in modulating the vagal tone and the structure properties of blood vessels.
Abstract 317: Endoplasmic Reticulum Stress Contributes to Aortic Stiffening Through Increasing Collagen Synthesis and Deposition
Authors: Spitler Kathryn , Wenceslau Camilla F , McCarthy Cameron G , Webb R. Clinton
Published: July 2021

Aortic stiffening is an independent predictor of negative cardiovascular outcomes and is associated with an increased risk of hypertension. Aortic fibrosis contributes to stiffening through the synthesis and deposition of collagen within the vessel wall. Endoplasmic reticulum (ER) stress has been linked with cardiac fibrosis and in non-vascular cell types ER stress can trigger collagen synthesis. However, the role of ER stress in aortic fibrosis and collagen synthesis remains unclear. We hypothesized that ER stress promotes aortic fibrosis through increasing collagen synthesis and deposition. In vitro, collagen synthesis was measured in the media from primary aortic vascular smooth muscle cells (VSMCs, third passage) treated at different time points (0, 6, 12, 24 hrs) with ER stress inducer, tunicamycin (TM, 1 μg/ml) using Sirius red and analyzed with a spectrophotometer (ODN 550 nM). In vivo, male Sprague-Dawley rats (12 wks) were implanted with mini-osmotic pumps containing TM (10 μg/kg/day, 14 days) or received sham surgery. Thoracic aorta was used for Western blot (data presented as fold change from control ± SEM) and immunohistochemical staining with Sirius red, a measure of collagen content and Masson’s trichrome stain, a measurement of fibrosis. Systolic blood pressure was increased in rats treated with TM compared to control (mmHg; 167±2 vs. 128±3). There was an increase in aortic wall thickness of the TM treated rats compared to control (mm; 0.35±0.01 vs. 0.32±0.01). Aortic expression of known proteins upregulated following induction of ER stress were increased by TM treatment: GRP78, an ER chaperone (1.4±0.3), PDI, a protein disulfide isomerase (1.3±0.2) and CHOP, a pro-apoptotic protein (1.7±0.3). Treatment with TM increased both aortic collagen content (% area; 10.7±0.5 vs. 7.4±0.4) and fibrosis (% area; 10.4±0.4 vs. 5.1±0.9) compared to control. There was a time-dependent increase in collagen synthesis from the media of VSMCs cells treated with TM (ODN 550nM: 0hrs, 0.9±0.002; 6hrs, 0.12±0.004; 12hrs, 0.17±0.005; 24hrs, 0.28±0.008). Our data suggest that ER stress may contribute to aortic stiffening through increasing collagen synthesis and deposition.
Abstract 363: Better Control of Blood Pressure in Midlife was Associated with Cognitive Function in Old Age: The Minabe Study in Japan
Authors: Iwahara Akihiko , Uematsu Yuji , Siba Mitsuru , Miyai Nobuyuki , Arita Mikio
Published: July 2021

Several studies show that hypertension in midlife is also related to cognitive impairment in old age. However, the associations between blood pressure and cognitive functions have been inconclusive. In this study, we examined this association in the sexagenarian and the septuagenarian separately to test our hypothesis that different risk factors were related to cognitive decline in each generation respectively. Participants were 1476 community-dwelling persons without dementia (708 men and 768 women). Resting systolic and diastolic blood pressures were measured in the seated position using an automated sphygmomanometer (HEM-907; Omron). The cognitive functions were measured by means of logical memory test, D-CAT (digit cancellation test; neuropsychological test for attention) and verbal fluency test. We asked participants about their use of antihypertensive drug and collected their data about blood pressure measured at a health examination conducted ten years ago. We constructed a series of multiple linear regression models to examine the association of blood pressure with each cognitive function. Separate analyses were conducted for each combination of predictor (SBP, DBP, past SBP, past DBP, and use of antihypertensive drug) and dependent value (logical memory test, D-CAT, and verbal fluency test). Regression models included the following covariates: age, education, gender, and body mass index. Results indicated the following two points: (1) Higher levels of past DBP (β = -.121, p < .05) was associated with decline in logical memory test and higher level of past SBP (β = -.127, p < .05) was associated with decline in verbal fluency test in the sexagenarian, (2) The use of antihypertensive drug was associated with decline in D-CAT (β = -.194, p < .001) and verbal fluency test (β = -.196, p < .05) in the septuagenarian. Our findings suggest that high blood pressure in the past decade is risk factor for cognitive decline in the sexagenarian but is not risk factor in the septuagenarian. The treatment of high blood pressure in midlife is important for the maintenance of cognitive functions. Early control of high blood pressure might reduce the risk of cognitive impairment in old age.
Abstract 377: Unravelling the Association between Blood Pressure and Insulin-like Growth Factor-1
Authors: Schutte Aletta E , Mels Catharina M , Smith Wayne , Schutte Rudolph , Malan Leone , Huisman Hugo W , Van Rooyen Johannes M , Fourie Catharina M , Malan Nico T , Kruger Ruan , Conti Elena
Published: July 2021

Conflicting reports exist on the relationship between systolic BP (SBP) and insulin-like growth factor-1 (IGF-1). Where positive associations are observed in patients with acromegaly, others find a negative association or no relationship. The versatile functions of IGF-1 as a growth factor (vasoprotective versus arterial and cardiac remodelling), the interplay with other hormones as well as IGF-1 resistance phenomena as those shown in chronic renal failure, are mostly regarded as confounding this relationship and thereby resulting in conflicting results. We aimed to review and compare regression coefficients for the association between SBP and total IGF-1 as reported in the literature and two studies from the general population.

We searched the literature for studies reporting the mentioned regression coefficients. We also included data from the SAfrEIC and SABPA studies in South Africa (total N=912) including African and Caucasian men and women, aged 20-70 yrs.

When plotting the regression coefficients from 13 studies with 20 data points against IGF-1 concentrations, we found a significant association between the regression coefficients and IGF-1 concentration (r=0.80; p<0.001). The slope intercepted the y-axis at IGF-1 concentration of 223.5 ± 14.4 ng/mL. Studies with mean IGF-1 at this range reported weak non-significant regression coefficients (typically young healthy men). Studies including acromegalic patients or adolescents reported significant positive associations, with negative associations for those with compromised cardiovascular function.

Our findings suggest that the regression coefficient for the SBP and IGF-1 relationship is dependent on the IGF-1 concentration. Positive associations are observed in situations with overtly high IGF-1 concentrations as those found in acromegaly or conditions of IGF-1 resistance, and beneficial negative relationships seen in those with low-normal IGF-1 and with compromised vascular function such as type 2 diabetes and hypertension. Our results suggest that the complex interplay between IGF-1 and other vasoactive hormones (e.g. growth hormone, insulin, cortisol and adipokines) or the development of a vascular resistance to IGF-1, may hamper the vasoprotective functions of IGF-1.
Abstract 388: ICV Angiotensin-(1-7) Improves Cardiac Function and Attenuates Remodeling In (mren2)27 Transgenic Hypertensive Rats
Authors: Kangussu Lucas M , Melo Marcos B , Guimarães Priscila S , Nadu Ana P , Bader Michael , Santos Robson A , Campagnole-Santos Maria J
Published: July 2021

Overactivity of the renin-angiotensin system (RAS), through Ang II/AT1 axis, plays a critical role in the pathogenesis of cardiovascular diseases. On the other hand, Ang-(1-7)/Mas axis is recognized to attenuate the deleterious effects of the RAS at different sites including the central nervous system. In the present study, Sprague Dawley (SD) and transgenic hypertensive rats (mRen2)27, instrumented with telemetry probes for arterial pressure (AP) measurements, were subjected to 14 days of lateral ventricle (ICV) infusion of Ang-(1-7) (200 ng/h) alone, Ang-(1-7) (200 ng/h) in association with a Mas receptor antagonist (A779, 1 μg/h) or saline (0.5 ml/hour) through osmotic mini-pumps. Ang-(1-7) ICV attenuated hypertension of (mRen2)27 rats (144±8 mmHg vs 174±3 mmHg, before. The AP lowering effect of ICV Ang-(1-7) was completely blocked by A779 (174±12 mmHg). Cardiac hypertrophy and dysfunction, evaluated by echocardiography, of (mRen2)27 rats were attenuated in Ang-(1-7) infused rats. The increased levels of atrial natriuretic peptide, brain natriuretic peptide, collagen I, fibronectin and TGF-β1 in the heart of (mRen2)27 rats were significantly reduced by Ang-(1-7) infusion and partially reversed by the concomitant infusion of A-779. Further, cardiac effects induced by ICV Ang-(1-7) were accompanied by an attenuation of the increased ratio sympathetic/ vagal activity to the heart of (mRen2)27. The data of the present study indicate that short-term central infusion of Ang-(1-7) produces Mas-mediate improvement in cardiac function and attenuates cardiac remodeling in (mRen2)27 hypertensive rats, probably through an improvement of the autonomic balance to the heart. Support: CAPES and FAPEMIG/CNPq through INCT-NanoBioFar and PRONEX.
Abstract 389: Elevation of Fatty Acid-Binding Protein 4 is Associated with Left Ventricular Hypertrophy and Diastolic Dysfunction in a General Population
Authors: Mita Tomohiro , Furuhashi Masato , Yuda Satoshi , Muranaka Atsuko , Ishimura Shutaro , Fuseya Takahiro , Akasaka Hiroshi , Yoshida Hideaki , Saitoh Shigeyuki , Miura Tetsuji
Published: July 2021

Fatty acid-binding protein 4 (FABP4/A-FABP/aP2) is expressed in both adipocytes and macrophages. Recent studies have shown secretion of FABP4 from adipocytes and association of elevated serum FABP4 level with obesity, insulin resistance, hypertension, and atherosclerosis. However, little is known about role of FABP4 in cardiac function. From database of the Tanno-Sobetsu Study, a study with prospective population-based cohort design, all data from female subjects who were on no medication and underwent echocardiography in 2011 (n = 108) were retrieved for analyses of relationships between serum FABP4 concentration, metabolic markers and parameters of echocardiography. FABP4 level was positively correlated with age, body mass index (BMI), blood pressure, triglycerides, HOMA-R, left ventricular (LV) wall thickness (LVWT; r = 0.39, p < 0.01), and E/e’ (r = 0.32, p < 0.01), an index of impaired LV relaxation, and negatively correlated with the ratio of early diastolic to atrial systolic velocity (E/A) of transmitral flow (r = -0.30, p < 0.01). FABP4 concentration was not significantly correlated with LV ejection fraction or LV diastole diameter. Multiple regression analysis adjusted by age and blood pressure revealed that FABP4 concentration was independently correlated with LVWT and E/e’. However, FABP4 level was not selected as an independent determinant of LVWT or E/e’ when BMI and/or HOMA-R were additionally incorporated into the adjustment. In conclusion, FABP4 may contribute to LV hypertrophy and diastolic dysfunction via facilitation of obesity and insulin resistance in the general population.
Abstract 405: Role of Adherent Platelets in 12-lipoxygenase-mediated Vascular Relaxation in Mouse Arteries
Authors: Kriska Tamas , Siangjong Lawan , Cepura Cody , Falck John R , Gauthier Kathryn M , Campbell William B
Published: July 2021

Lipoxygenase (LO) metabolites of arachidonic acid (AA) regulate vascular tone in mice. Previously, vasoactive LO metabolites such as 11,12,15-trihydroxy-eicosatrienoic acid or 12(S)-hydroxy-eicosatetraenoic acid (12-HETE) were identified in murine arteries. However, the specific LO responsible for the synthesis of these metabolites was not found in vascular endothelium or smooth muscle. The synthesis of 12-LO metabolites depended on the mode of sample preparation. The synthesis of [14C]-AA metabolites was dramatically increased in buffer rinsed, control aortas when compared to heparin-rinsed aortas. We hypothesized that adherent platelets (PLs) are responsible for the synthesis of vasoactive 12-LO metabolites in mouse arteries. Aortic PL adherence and removal of PLs by heparin rinsing were confirmed by immunohistochemical and western immunoblot analysis using specific anti-PL-12-LO and gpIIb/IIIa antibodies. In the presence of indomethacin and L-NA, acetylcholine caused concentration-dependent relaxations in rinsed, control abdominal aortic rings that were preconstricted with thromboxane-mimetic,
U46619
(maximum relaxation at 86.4±3.6%) or phenylephrine (maximum relaxation at 81.7±4.1%); however, those relaxations were significantly reduced in heparin-rinsed aortas to 71.6±4.5% (p<0.05) and 48.3±7.2% (p<0.005), respectively. Isolated PLs and rinsed, control aortas showed identical [14C]-AA metabolic patterns. Mass spectrometric identification of AA metabolites produced by rinsed, control aortas or isolated PLs revealed an identical composition consisting of 12-HETE, trioxilins A3 and C3 and hepoxilin A3. Synthetic trioxilin A3, trioxilin C3 and hepoxilin A3 (3 μM) relaxed
U46619
-preconstricted mesenteric arteries with maximum relaxations of 78.9±3.2; 82.2±5.0 and 20.0±5.1, respectively. These data indicate that adherent PLs contribute to vascular relaxation by producing PL-12-LO-derived relaxing factors. This effect should be taken into consideration when performing functional studies in isolated mouse arteries. Heparin rinsing of isolated arteries is important to remove adherent PLs and eliminate their contributions to vascular tone.
Abstract 432: Up-regulation of Calcyon Increases Arterial Pressure via Enhancement of Adrenergic Transmission
Authors: Elmarakby Ahmed A , Vazdarjanova Almira , Bergson Clare
Published: July 2021

Recent studies suggest a link between blood pressure (BP) and executive functions including working memory, response inhibition, and attention. Older adults and children with hypertension exhibit deficits in working memory and attention; however, it is not clear whether a common mechanism could link the elevation in BP and the cognitive abnormalities. The expression of the Calcyon (Caly), a protein which regulates receptor endocytosis, is increased in the brain of the spontaneously hypertensive rat (SHR), a widely accepted animal model of essential hypertension and attention deficit hyperactivity disorder. We hypothesize that Caly up-regulation in forebrain elevates BP under normal physiological response by altering adrenergic transmission. Radio-telemetry transmitters were implanted in CalOE transgenic mice in which Caly is up-regulated, and in tTA littermate controls. Mean arterial pressure was significantly higher in CalOE mice compared to tTA control (121± 1 vs. 108±1 mmHg, P<0.05). Consistent with deficits in executive functions, CalOE mice were also more active and less able to extinguish a learned behavior than tTA controls. Silencing the transgene with doxycycline treatment improved learning deficits and prevented the elevation in BP in CalOE mice. Plasma nor-epinephrine levels were higher in CalOE vs. tTA control mice (20±2 vs. 13±4 ng/ml). In the brain, dopamine levels were significantly lower in CalOE mice vs. control (0.2 ± 0.02 vs. 0.5±0.08 ng/mg) together with decreased dopamine D1 receptor expression whereas urinary dopamine excretion levels were higher in CalOE vs. control (0.5± 0.1 vs. 0.1± .04 μg/day, P<0.05). These data suggest that up-regulation of Caly in brain could increase BP and compromise inhibitory control mechanisms under normal physiological condition via the differential regulation of adrenergic transmission.
Abstract 434: Increased Dietary Salt Intake Enhances the Exercise Pressor Reflex
Authors: Yamauchi Katsuya , Tsuchimochi Hirotsugu , Stocker Sean D , Kaufman Marc P
Published: July 2021

Increased dietary salt in rats has been shown to sensitize central sympathetic circuits and enhance sympathetic responses to several stressors, including hyperinsulinemia, intracerebroventricular injection of angiotensin and electrical stimulation of sciatic nerve afferents. These findings prompted us to test the hypothesis that increased dietary salt enhanced the exercise pressor reflex.Male Sprague-Dawley rats were fed 0.1% (n=16) or 4.0% (n=18) NaCl chow for 14-21 days. Then, rats were decerebrated and the exercise pressor reflex was produced by static contraction of the hindlimb muscles through electrical stimulation of the cut peripheral ends of the L4 and L5 ventral roots. We found that contraction produced a significantly greater increase in mean arterial pressure of rats fed 4.0% NaCl (24±2 mmHg) versus 0.1% NaCl (15±2 mmHg). Baseline mean arterial pressure was not different between 0.1% versus 4.0% NaCl diets (79±5 vs 83±4 mmHg). The tension time indices were not different between the 0.1% (17±1 kg per s) versus 4.0% (18.2±2 kg s) NaCl groups (P = 0.422). The enhanced responsiveness to contraction appeared to be attributable to sensitized group III mechanoreceptors because the renal and lumbar sympathetic responses to contraction were augmented within 200 ms of the onset of contraction. Section of the L4 and L5 dorsal roots abolished the pressor responses to contraction in both groups of rats, an effect showing that the responses were reflex in origin. We conclude that increased dietary salt can enhance the reflex sympathetic responses to static exercise.
Abstract 444: Presence and Functions of B1- and B2-adrenergic Receptors on Bulbospinal Neurons in the Rostral Ventrolateral Medulla
Authors: Oshima Naoki , Onimaru Hiroshi , Yamamoto Kojiro , Takechi Hanako , Nishida Yasuhiro , Oda Takashi , Kumagai Hiroo
Published: July 2021

The presence and effects of β-adrenergic receptors (β-ARs) on bulbospinal RVLM neurons are little examined. To directly investigate whether RVLM neurons have sensitivity to propranolol (a non-selective β-AR antagonist), metoprolol (a β1-AR antagonist), dobutamine (a β1-AR agonist), butoxamine (a β2-AR antagonist), and salbutamol (a β2-AR agonist), we examined changes in the membrane potentials (MPs) of bulbospinal RVLM neurons using whole-cell patch-clamp technique, during superfusion with these drugs. During propranolol superfusion eight of 17 RVLM neurons were depolarized, and nine of 17 RVLM neurons were hyperpolarized. In contrast, during metoprolol superfusion sixteen of 20 RVLM neurons were hyperpolarized, and during dobutamine superfusion five of 6 RVLM neurons were depolarized. During butoxamine superfusion eleven of 16 RVLM neurons were depolarized, and during salbutamol uperfusion all of 8 RVLM neurons were hyperpolarized. To clarify the character of RVLM neurons by themselves, superfusion with metoprolol or butoxamine, dissolved in a low-Ca
2+
, high-Mg
2+
solution, was performed. During metoprolol superfusion nine of 12 RVLM neurons were again hyperpolarized, and during butoxamine superfusion seven of 8 RVLM neurons were depolarized. These results suggest that β1- and β2-ARs are actually present on the RVLM neurons. β1-AR antagonists decreased the activity of bulbospinal RVLM neurons, and β2-AR antagonists increased it. To determine the presence of β1- and β2-ARs histologically, immunofluorescent examination including double staining was performed. All of 5 metoprolol-hyperpolarized neurons showed β1-AR immunoreactivity, and all of 3 butoxamine-depolarized neurons showed β2-AR immunoreactivity. Furthermore, two RVLM neurons, which were depolarized during metoprolol superfusion and were hyperpolarized during followed butoxamine superfusion, were examined for β1- and β2-ARs immunoreactivity. These neurons showed immunoreactivity for both β1- and β2-ARs, and most β1-ARs immunoreactive neurons in the RVLM had immunoreactivity for β2-ARs.Our findings suggest that β1-AR antagonists or β2-AR agonists may decrease blood pressure through decreasing activity of bulbospinal RVLM neurons.
Abstract 446: Rostral Ventrolateral Lateral Medulla Mediates the Sympathetic and Cardiovascular Response to Increased Cerebrospinal Fluid Sodium Concentration
Authors: Stocker Sean D
Published: July 2021

Compelling evidence indicates increased cererbrospinal fluid (CSF) sodium elevates sympathetic nerve activity (SNA) and arterial blood pressure (ABP) in salt-sensitive hypertension. RVLM neurons project to the spinal cord and regulate SNA and ABP under a number of physiological challenges and pathophysiological states. Therefore, we hypothesized that RVLM neurons mediate the sympathoexcitatory response to increased CSF sodium. Inactin-anesthetized, male Sprague-Dawley rats (n=5/group) were prepared for sympathetic nerve recordings and a lateral ventricle brain cannula. Infusion of 1M NaCl (5 μL/10 min) to increase CSF sodium concentrations by 5mM produced a significant (P’s<0.05, n=6) increase in lumbar SNA (Δ: 115±3%), adrenal SNA (Δ: 122±3%), and mean arterial blood pressure (Δ: 8±1 mmHg). The infusion did not affect splanchnic SNA (Δ: 102±4%) but decreased renal SNA (Δ: 91±2%). Inhibition of the RVLM with bilateral injection of the GABA agonist muscimol (2.5mM per 50 nL per side) significantly (P’s<0.05; n=5) attenuated the increased lumbar SNA (Δ:101±2%), adrenal SNA (Δ:105±2%), and mean arterial blood pressure (Δ: 1±1mmHg, P’s<0.05; n=6). Blockade of ionotropic glutamate receptors in the RVLM with bilateral injection of kynurenic acid (30mM per 50 nL per side) also significantly (P’s<0.05; n=5) attenuated the increase in lumbar SNA (Δ: 101±3%), adrenal SNA (Δ: 110±3%) and mean ABP (Δ: 1±2 mmHg) to lateral ventricle infusion of 1M NaCl (5uL/10 min). In a final set of experiments, in vivo single-unit recordings demonstrate that ventricular infusion of 1M NaCl (5uL per 10 min) increased discharge in 60% (6/10) of spinally-projecting, barosensitive RVLM neurons (2.1±0.4 to 5.8±0.2 Hz, P<0.05). Infusion of artificial CSF did not affect any variable. These findings suggest increased CSF sodium activates a glutamatergic pathway to RVLM neurons to elevate SNA and ABP.