bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 354: Cardiovascular Responses to Chronic Baroreflex Activation in Aldosterone Hypertension
Authors: Lohmeier Thomas , Liu Boshen , Georgakopoulos Dimitrios , Cates Adam , Irwin Eric
Published: March 2022

Both suppression of sympathetic activity (SA) by chronic electrical activation of the carotid baroreflex (BA) and renal denervation (RDX) abolish obesity-induced hypertension (HT), which is mediated by increased SA. Evidence in support of a sympathetic component to aldosterone (Aldo) HT is equivocal. Therefore, at the same stimulation parameters in each dog, cardiovascular responses to 7 days of BA were determined in 9 dogs before (no HT) and after induction of HT by infusion of Aldo for 14 days (12μg/kg/day), which increased plasma levels ~ 10-fold (control= 4.3±0.4 ng/dL). 3 dogs with Aldo HT were subjected to RDX. Aldo HT was associated with increases in plasma osmolality (293±1 to 297±1 mOsm/kg) and [vasopressin] (AVP, Table), marked polydipsia (241± 90 to 2,380 ± 382 mL/day), hypokalemia (4.2±0.1 to 2.3±0.1 mmol/L), and suppression of plasma renin activity, but no significant changes in plasma [norepinephrine] (NE, Table). MAP (mmHg), NE (pg/mL), and AVP (pg/mL) responses to BA:

*P<0.05 vs Control

Plasma [NE] decreased with BA during both no HT and Aldo HT, indicating suppression of SA, but MAP lowering was appreciably diminished during Aldo HT. In contrast, there was no lowering of MAP after RDX. There were no significant changes in plasma osmolality, drinking, or [AVP] during BA. These findings indicate that classic renal mineralocorticoid mechanisms, not increased SA, account for the initial progression of HT produced by pathophysiological circulating levels of Aldo. Further, they show that chronic stimulation of baroreceptors afferents has differential central effects to suppress SA without altering control of plasma osmolality by suppressing thirst or AVP secretion.
Abstract 357: Role of Blood Pressure in Aldosterone-Mediated Cardiac Injury
Authors: Ball Jana P , Syed Maryam , Maranon Rodrigo O , Reckelhoff Jane F , Yanes Licy L , Romero Damian G
Published: March 2022

Excess aldosterone (ALDO) causes hypertension (HTN), and cardiac hypertrophy, injury and dysfunction. The role of HTN in ALDO-mediated cardiac injury remains controversial. Whether cardiac injury is blood pressure (BP)-dependent, -independent or exacerbated by HTN is not fully understood. We aim to study the role of BP in ALDO-mediated cardiac hypertrophy, inflammation and fibrosis.

Eight-week old uninephrectomized male Sprague Dawley rats (N=6/grp) were randomly assigned to the following 3 groups: Control, ALDO/SALT, ALDO/SALT+3-AHT (Triple Anti-Hypertensive Therapy). ALDO (0.75 μg/h) or vehicle (PEG 300) was administered by osmotic minipumps. SALT (1.0 % NaCl + 0.3 % KCl) was administered in tap water. 3-AHT (240 mg/kg hydralazine, 75 mg/kg hydrochlorothiazide, 15 mg/kg reserpine) was administered in the food. Another set of animals (N=6/grp) was implanted with radiotelemetry probes for BP monitoring. Left ventricle (LV) gene expression (GE) was quantified by qRT-PCR.

3-AHT reduced MAP (126 ± 4 vs. 178 ± 3 mm Hg, p<0.05 vs. ALDO, controls: 108 ± 1). 3-AHT attenuated cardiac (3.8 ± 0.1 vs. 4.0 ± 0.1 HW/BW, p<0.05 vs. ALDO; controls: 3.5 ± 0.1) and LV (2.8 ± 0.1 vs. 3.0 ± 0.1 LVW/BW, p<0.05 vs. ALDO; controls: 2.4 ± 0.1) hypertrophy. 3-AHT abolished ALDO-mediated LV GE upregulation of fibrosis (collagen I, collagen III, fibronectin, Timp1, periostin) and inflammation (osteopontin, MCP-1, TGFβ, Hmox1) markers. A similar effect was observed with novel LV ALDO-target genes such as Reg3b, collagen VIII, Prss35, Ltbp2 or Pcp4. 3-AHT drug administration in the food caused no change in NaCl intake. However, to avoid the confounding effect of the natriuretic drug hydrochlorothiazide, a group of animals was treated with hydralazine and reserpine alone (2-AHT). 2-AHT treatment attenuated HTN (135 ± 4 vs. 178 ± 3 mm Hg, p<0.05 vs. ALDO) but did not modify ALDO-mediated cardiac or LV hypertrophy. However, 2-AHT also abolished ALDO-mediated GE upregulation of fibrosis and inflammation markers, as well as novel LV ALDO-target genes.

These results suggest that ALDO-mediated LV GE regulation in vivo is BP-dependent without excluding that ALDO may exacerbate the HTN-mediated cardiac injury. A stricter BP management may benefit patients with elevated ALDO levels.
Abstract 366: Midodrine is Associated With a Lower Risk of All-cause Hospitalizations Compared With Fludrocortisone in Patients With Orthostatic Hypotension and Heart Failure
Authors: Shibao Cyndya , Grijalva Carlos G , Lipsitz Lewis A , Biaggioni Italo , Griffin Marie R
Published: March 2022

Orthostatic hypotension (OH) causes approximately 80,000 hospitalizations per year in the US. Current treatment for OH includes midodrine and fludrocortisone. Although these medications have been

used for decades for treatment of OH, their relative effectiveness and safety is unknown. We compared incidence rates of hospitalizations for all causes, for congestive heart failure (CHF, safety outcome) and for syncope (effectiveness outcome) among users of midodrine compared to users of fludrocortisone (reference) in a retrospective cohort study of Tennessee Medicaid enrollees (1996-2008). Adjusted rate ratios were

calculated using propensity scores. We studied 636 patients initiating midodrine and 1066 patients initiating fludrocortisone. Incidence rates of all-cause hospitalizations for midodrine and fludrocortisone users were 1398 and 1515 per 1,000 person-years, respectively (adjusted incidence-rate ratio (aIRR) 0.88, 95% CI (0.75-1.03). The respective rates of syncope-related hospitalization were 129 and 129 per 1,000 person-years (aIRR: 0.90, 95% CI:0.60-1.36). Among 452 (27%) patients with prevalent CHF, the rates of all-cause hospitalization were 1786 and 2413 per 1000 person-years (aIRR: 0.66, 95% CI:0.47-0.87) (Figure), and the respective rates of CHF exacerbation-related hospitalizations were 285 and 297 per 1,000 person-years (aIRR: 0.60, 95% CI:0.34-1.06). Compared with fludrocortisone, users of midodrine had similar rates of all-cause and syncope-related hospitalizations. However, among patients with CHF, midodrine use was associated with a lower risk of hospitalization.
Abstract 368: Insulin Promotes Hypoxic Vascular Injury in Insulin Resistance
Authors: Katakam Prasad V , Liu Dan , Rutkai Ibolya , Gordon Angellica O , Grovenburg Samuel M , Dutta Somhrita , Busija David W
Published: March 2022

Clinical studies targeting aggressive lowering of blood glucose in type 2 diabetes (T2DM) by exogenous insulin failed to show cardiovascular protection, but paradoxically increased cardiovascular mortality. Previously, we demonstrated cerebrovascular insulin resistance (IR) and reduced mitochondria-mediated vasodilation in Zucker obese (ZO) rats with IR compared to lean (ZL) controls. Communication between endoplasmic reticula (ER) and mitochondria play a critical role in metabolic functioning and responses to hypoxia. Tethering of mitochondria to ER by mitofusin 2 (MFN2) creates Ca
2+
microdomains to aid inter-organelle communication. Overexpression of glucose regulated protein 78 kDa (GRP78), an ER chaperone and a marker of ER stress, is protective by preempting ER Ca
2+
leak. Our aim is to demonstrate the adverse impact of IR on ER-mitochondrial interactions. Levels of mitochondrial proteins (MFN2 and voltage dependent anionic channel or VDAC), ER stress marker (GRP78), and endothelial nitric oxide synthase (eNOS) were determined in cerebral arteries from ZO and ZL treated with vehicle or insulin under normoxic or hypoxia-reoxygenation (H-R) conditions. ZO arteries displayed decreased MFN2 and VDAC, elevated GRP78, and increased eNOS monomer/dimer ratio (eNOS uncoupling) at baseline. In ZL arteries, MFN2 and GRP78 proteins were increased under normoxic (1.2±0.1 and 0.97±0.27 respectively; p<0.05 versus vehicle) and decreased under H-R conditions (1.43±0.4 and 0.43±0.02 respectively; p<0.05 versus vehicle) by insulin. In contrast, in ZO arteries, MFN2 and GRP78 proteins were decreased under normoxic (0.69±0.1 and 0.71±0.01 respectively; p<0.05 versus vehicle) and increased under H-R conditions (1.76±0.12 and 1.4±0.07 respectively; p<0.05 versus vehicle) by insulin. Insulin decreased eNOS uncoupling under H-R versus normoxia in ZL, but increased it in ZO arteries. Thus, ZO arteries with IR display impaired mitochondria-ER communication at baseline which was further exacerbated by insulin under H-R leading to increased oxidative vascular injury. Our study provides a mechanistic basis for increased cardiovascular mortality following intensive glucose lowering therapy by high exogenous insulin in patients with IR and T2DM.
Abstract 384: Tumor Necrosis Factor-alpha Antagonism Attenuates Hypertension and Renal Injury Induced by Chronic Blockade of Nitric Oxide and High Salt Intake in Mice
Authors: Singh Purnima , Castillo Alexander , Prieto Minolfa C , Majid Dewan S
Published: March 2022

Earlier, we demonstrated that systemic blockade of nitric oxide (NO) generation by acute infusion of nitro-L-arginine methyl ester (L-NAME) in mice increases plasma and renal levels of tumor necrosis factor-alpha (TNF-α). In the present study, we examined the role of TNF-α in mediating hypertension and renal injury responses to chronic NO blockade by evaluating the responses to chronic administration of L-NAME (0.05 μg/g/min; by osmotic mini-pump) for 3 wks in mice (C57BL6) treated with or without TNF-α receptor antagonist, etanercept (ETN; 5 mg, ip, every alternate day). These mice were given either normal (NS; 0.03% NaCl) or high salt (HS; 4% NaCl) containing diets. Systemic blood pressure (BP) was measured by tail-cuff plethysmography. At the end of the experimental period, mice were sacrificed to collect plasma and renal tissue samples to measure TNF-α level by ELISA and to determine the extent of tissue injury such as glomerulosclerosis (GS; PAS staining) and renal interstitial fibrosis (RIF; Trichrome staining). L-NAME treatment caused increase in BP which was greater in HS (104±3 to 139±3 mmHg; n=6) than in NS group (96±6 to 124±6 mmHg; n=6). ETN treatment significantly attenuated this increase in BP in both L-NAME+HS (119±11 mmHg; n=6) and in L-NAME+NS (115±8 mmHg, n=6) groups. Renal tissue level of TNF-α was higher in L-NAME+NS (664±100 vs 325±73 pg/mg protein) and in L-NAME+HS (391±38 vs 114±17 pg/mg protein) groups compared to the levels in groups given NS (n=5) or HS (n=5) diet alone. However, the changes in plasma level of TNF-α due to chronic L-NAME treatment were minimal. ETN treatment lowered renal TNF-α level in L-NAME+NS (356±60 pg/mg protein) and in L-NAME+HS (259±65 pg/mg protein) groups. L-NAME treatment significantly increased GS (7.1±1.0 vs 17.9±2.3 % sclerosis area) and RIF, (1.5±0.1 vs 5.8±0.6 % fibrotic area) in HS group. However, these changes were minimal in NS group (GS, 10.3±1.0 vs 13.2±1.5 % and RIF, 1.3±0.1 vs 2.4±0.3 %). ETN treatment markedly attenuated the GS (11.8±0.4% and 11.9±0.6 %) and RIF (2.6±0.3% and 1.9±0.3%) both in L-NAME+HS and in L-NAME+NS groups. These data demonstrate that an enhancement in TNF-α activity is involved in mediating hypertension and renal injury responses to chronic NO blockade and HS intake.
Abstract 390: Micro RNA-499 Protects the Heart Against Adverse Post Myocardial Infarction Remodeling and Regenerates Damaged Myocardium by Promoting Endogenous Cardiac Stem Cell Differentiation
Authors: Givvimani Srikanth , Kalani Anuradha , Narayanan Nithya , Kundu Sourav , Tyagi Suresh
Published: March 2022

The intrinsic capacity of the heart to regenerate and replace damaged myocardium by promoting cardiac stem cell differentiation is one of the key repair mechanisms following acute injury to heart. Mitochondrial dysfunction plays an important role in various heart disorders including myocardial infarction. Micro RNAs are implicated in pathogenesis of various cardiac diseases. Cardiac specific miR-499 was shown to regulate mitochondrial fission by inhibiting Drp-1 (Dynamin-related protein). We hypothesize that mitochondrial division inhibitor (Mdivi-1) ameliorates adverse post myocardial infarction remodeling by increasing the expression of miR-499 thereby promoting the ventricular cardiac stem cell differentiation . To verify this, we created myocardial infarction in wild type (C57BL6/J) animals and treated them with Mdivi-1(50mg/kg BW) for 4 weeks. Cardiac function was assessed by echocardiography and pressure volume loop. Individual micro RNA assay was performed to compare miR-499 expression among the groups. Endogenous cardiac stem cell expression was assessed by C-kit marker. Our results showed that Mdivi-1 promotes miR-499 expression and cardiac stem cell differentiation. Echo data revealed improved ejection fraction and left ventricular function. Based on these findings we conclude that Mdivi-1 ameliorates adverse post myocardial infarction remodeling by promoting miR-499 expression and cardiac stem cell differentiation.
Abstract 4: Transient Receptor Potential Melastatin 7 Cation Channel (TRPM7) Kinase Domain - a New Player in Angiotensin II-induced Hypertension and Cardiac Hypertrophy
Authors: Antunes Tayze T , Callera Glaucia E , He Ying , Ryazanov Alexey G , Ryazanova Lilian V , Touyz Rhian M
Published: March 2022

Transient receptor potential melastatin 7 (TRPM7) cation channel is a unique protein that has the dual ability to act as a channel to regulate transmembrane Mg
2+
transport and also as a kinase to promote cellular signaling. Despite increasing awareness of the importance of Mg
2+
in cardiovascular biology nothing is known about TRPM7 and its kinase domain in the pathophysiology of hypertension. We previously demonstrated that Ang II regulates TRPM7
in
vitro
. Here we studied TRPM7 kinase-deficient mice to explore the role of the TRPM7 kinase domain in Ang II-induced hypertension. TRPM7 kinase deficient mice (TRPM7
+/-
) and wild type (WT) counterparts were infused with Ang II (400 ng/kg/min; minipumps) for 4 weeks. Blood pressure (BP) was measured by tail cuff. Vascular reactivity and structure studies were performed by myography in mesenteric arteries. Although baseline BP tended to be higher in TRPM7
+/-
versus WT mice (127 ± 6.0 vs 119 ± 2.2 mmHg), significance was not achieved. TRPM7
+/-
mice displayed earlier onset of BP increase by Ang II (2 weeks; WT-Ang II: 145 ± 5 vs TRPM7
+/-
Ang II: 178 ± 9; mmHg). After 4 weeks, BP was significantly higher in TRPM7
+/-
(174 ± 10 mmHg) than in WT mice (147 ± 8 mmHg). Ang II-induced hypertension was associated with cardiac hypertrophy, an effect that was exaggerated in TRPM7
+/-
mice (WT: 4.4 ± 0.1; WT-Ang II: 5.0 ± 0.2; TRPM7
+/-
: 4.5 ± 0.1; TRPM7
+/-
Ang II: 5.7 ± 0.1 g/body weight). Mesenteric arteries from Ang II-infused TRPM7
+/-
mice exhibited decreased sensitivity to acetylcholine (pD2; WT-Ang II: 7.6 ± 0.3 vs. TRPM7
+/-
Ang II: 6.7 ± 0.4), and reduced maximal relaxation compared to WT mice (WT-Ang II: 88 ± 8% vs TRPM7
+/-
Ang II: 59 ± 10%). Ang II induced a leftward shift in the stress-strain relationship for both WT and TRPM7
+/-
mice in a similar fashion. Plasma analysis revealed that TRPM7
+/-
mice were hypomagnesemic, and that Ang II increased Mg
2+
levels to a greater extent in WT than in TRPM7
+/-
mice (WT: 0.65 ± 0.02; WT-Ang II: 0.74 ± 0.04; TRPM7
+/-
: 0.60 ± 0.01; TRPM7
+/-
Ang II: 0.64 ± 0.02; mmol/L). In conclusion, our findings demonstrate that hypertension, cardiac hypertrophy and endothelial dysfunction are exaggerated by Ang II in TRPM7
+/-
hypomagnesemic mice, suggesting a novel role for TRPM7 kinase domain in cardiovascular pathophysiology.
Abstract 402: Effect of Aging on Endothelial Function in a Genetic Mouse Model of Hypertension
Authors: Didion Sean P
Published: March 2022

Although previous studies have demonstrated that there is a strong genetic component associated with the development of hypertension, angiotensin II-infusion represents the most commonly used model of hypertension, especially in mice. While the BPH/2 mouse represents a genetic, non-angiotensin II-dependent, model of hypertension, there is limited information regarding endothelial responses and, to our knowledge, no information regarding the effect of aging on blood pressure or endothelial function in this model. The goal of this study was to test the hypothesis that endothelial dysfunction is present in the carotid artery in the genetically hypertensive BPH/2 mouse and that the degree of endothelial dysfunction in this model worsens with age. Systolic blood pressure (SBP) and vascular function was examined in young (5 months) and aged (12 months of age) BPN/3 and BPH/2 mice. SBP was significantly greater (P<0.05) in young hypertensive mice as compared to their normotensive counterparts (e.g., SBP was 151±6 and 122±3 mmHg in BPH/2 and BPN/3 mice, respectively). Blood pressure was not affected by age as SBP averaged 147±3 and 127±3 mmHg in aged BPH/2 and BPN/3 mice, respectively. Acetylcholine-induced relaxation was impaired by approximately 30% (P<0.05) in BPH/2 mice as compared to BPN/3 mice (e.g., 10 μM acetylcholine produced 65±6 and 90±5% relaxation in BPH/2 and BPN/3 mice, respectively). Moreover, the degree of endothelial dysfunction was markedly greater (P<0.05) in aged as compared to young BPH/2 mice. Tempol, a scavenger of superoxide, was very effective in improving endothelial responses in both young and aged BPH/2 mice, suggesting an important role for superoxide in the impairment of endothelial function. Impairment of acetylcholine-induced relaxation was selective for endothelium as responses to nitroprusside were not altered in BPH/2 mice and unaffected by age. These data demonstrate that endothelial dysfunction is present in the carotid artery of BPH/2 mice and is mediated in large part by superoxide. Aging appears to be associated with the worsening of the vascular phenotype and as such BPH/2 mice represent a novel genetic mouse model of hypertension-related, aging-induced endothelial dysfunction. NIH
HL089884
&
HL107632
Abstract 406: Improved Angiotensin II Type 2 Receptor Expression and Function in Transglutaminase 2 Knock-Out Mice Treated with Angiotensin II
Authors: Savoia Carmine , Arrabito Emanuele , Sada Lidia , Michelini Serena , Pucci Lorenzo , Briani Martina , Nicoletti Carmine , Candi Eleonora , Steckelings Ulrike M , Schiffrin Ernesto L , Volpe Massimo
Published: March 2022

Objective:
Transglutaminase 2 (TG2) induces transamidation and cross-linking of proteins that can modulate receptor interaction. Angiotensin II (AngII) may regulate TG2 and AngII type 2 receptor (AT2) expression and function. We hypothesized that AngII may impair AT2 expression and function through TG2.

Methods:
TG2-knockout mice (TG2-K/O, 8 weeks old, 6 for each group) and age-matched wild type (WT) mice were treated or not with AngII (400 ng/kg/min) for 14 days. Vascular reactivity was assessed in response to sodium nitroprusside (SNP, 10 nM to 1 mM), in mesenteric arteries pre-contracted with norepinephrine (10 μM). AT2 function was assessed by concentration-response curve to the selective AT2 agonist Compound 21 (C21, 1nM to 1 μM) in mesenteric arteries pre-contracted with norepinephrine. AT2 expression in aorta was evaluated by immunoblotting.

Results:
C21-induced relaxation was similar in untreated WT, and in untreated TG2-K/O. C21-induced relaxation was improved only in AngII-treated TG2-K/O (2-fold increase vs untreated TG2-K/O, P<0.001). SNP dependent relaxation was similar in all groups. AT2 receptor expression was similar in untreated WT and untreated TG2-K/O. AT2 was reduced by AngII in WT (-36±6% vs untreated WT, P<0.01), and significantly increased in TG2-K/O (+53±4% vs untreated TG2-K/O, P<0.001).

Conclusions:
AngII fails to reduce AT2 expression and function in TG2-K/O mice. Therefore, AngII may negatively modulate AT2 receptor expression and function through TG2.
Abstract 407: Tyramine Reveals a Functional Adrenergic System in Perivascular Adipose Tissue
Authors: Watts Stephanie W , Burnett Robert , Ayala-Lopez Nadia , Mahon Bridget , Dorrance Anne M , Jackson William F , Fink Gregory D
Published: March 2022

The sympathetic nervous system is undeniably important to blood pressure control. With the discovery of an adrenergic system in perivascular adipose tissue (PVAT) around both the aorta and mesenteric artery, we hypothesized that this system could promote contraction of the vessel around which it lives. Using isolated tissue bath and HPLC techniques, we examined the mechanism of contraction to the indirect sympathomimetic tyramine. Tyramine (100 nM - 1 mM) caused a concentration dependent contraction that was dependent on the presence of PVAT in normal rat thoracic aorta (figure, maximum tyramine contraction as a % initial phenylephrine contraction + PVAT = 47+/2, -PVAT = 11+/2), superior mesenteric artery (+ PVAT =64+/15 , -PVAT = 6+/2), and mesenteric resistance artery (+ PVAT =83+/17 , -PVAT = 3+/1). Tyramine stimulated release of NE, dopamine (DA) and serotonin (5-HT) from PVAT isolated from the superior mesenteric artery. In the thoracic aorta, tyramine induced contraction (Max = 61+/-13%) was rightward shifted and maximum contraction inhibited by the norepinephrine transporter inhibitor nisoxetine (1 uM =25+/-9%), vesicular monoamine transporter tetrabenazine (10 uM; 43+/-10%) and abolished by the alpha adrenergic receptor antagonist prazosin (100 nM). Removal of the celiac ganglion as a neuronal source of NE reduced PVAT content by ~38% and reduced maximum tyramine contraction by only ~22%. These data suggest that PVAT components that are independent of the formal SNS can release NE in a tyramine-sensitive manner. One of these components may be the adipocyte itself, a novel finding that has implications for control of vascular tone and local metabolic control.
Abstract 408: Severity of Hypertension does not Correlate with Increased miR-221 and miR-222 Expression
Authors: Woods T C
Published: March 2022

Multiple smooth muscle microRNA (miR) are required for normal regulation of blood pressure. miR-221 and miR-222 promote vascular remodeling through down regulation of the cyclin-dependent kinase inhibitor, p27Kip1. The vasculature of the spontaneously hypertensive rat (SHR) exhibits reduced expression of p27Kip1 that promotes vascular remodeling. Internal mammary arteries (IMAs) of diabetic subjects undergoing coronary artery bypass grafting exhibit elevated levels of miR-221 and miR-222. Given the relationship between diabetes and hypertension and the fact that the SHR exhibit low levels of p27Kip1, we hypothesized that increasing severity of hypertension would correlate with an increase in miR-221 and miR-222 in the vasculature that promotes vascular remodeling. Using multivariate linear regression, we evaluated the levels of miR-221 and miR-222 in the IMAs of both diabetic (n=19) and non-diabetic (n=18) subjects with reference to systolic and diastolic blood pressure, number of anti-hypertensive medications, and whether hypertension was controlled. Additionally, we tested whether individual anti-hypertension medication classes correlated with changes in miR-221 and miR-222 levels. When corrected for the presence of diabetes, none of these parameters correlated with a change in miR-221 and miR-222 levels. These data suggest both that miR-221 and miR-222 are not among the smooth muscle miRs involved in the regulation of blood pressure, and that hypertension does not promote vascular remodeling through changes in miR-221 and miR-222.
Abstract 415: Identification of Intracellular Signaling Pathways in Human Endothelial Cells Stimulated by Angiotensin-(1-7)
Authors: Meinert Christian , Gembardt Florian , Greenberg Barry , Walther Thomas
Published: March 2022

Despite many publications regarding the physiological actions of Ang-(1-7) and its beneficial effects under pathophysiological circumstances, the intracellular signaling initiated by Ang-(1-7) remains still elusive. It was the aim of our study to identify proteins regulated by Ang-(1-7) and to determine potential intracellular signaling cascades.

Human Umbilical Vein Endothelial Cells (HUVEC) were stimulated either with 10
-7
M Ang-(1-7) or the solvent for 1h, 3h, 6h, and 9h. Effects of Ang-(1-7) on intracellular signaling were assessed via protein microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify potential intracellular Ang-(1-7) signaling pathways. Protein microarray data has been verified by Western blot and Real-Time PCR.

The microarray identified 107 regulated proteins after 1h, 139 after 3h, 33 after 6h, and 77 after 9h. Regulated proteins could be associated with high significance to metabolic pathways like “Molecular Mechanism of Cancer” and “Chronic Myeloid Leukemia” signaling, the latter fitting our recent findings that Ang-(1-7) stimulates hematopoetic progenitor cells. The regulation of identified proteins fitted into the signaling cascades in a time dependent manner. Western blot results for PIAS2 (early protein) were in accordance to the protein microarray data and displayed a decrease by almost 45% after Ang-(1-7) stimulation for 1h and 3h. Additionally, we were able to verify the protein microarray results for FBI-1/Pokemon (late protein) after stimulation for 9h, but not after 6h. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types like HDMEC and bEnd.3. Further experiments showed that PIAS2 protein was affected by Ang-(1-7) already after 30 min, whereby Real-Time PCR showed no influence of Ang-(1-7) on mRNA level.

Our experiments identified new proteins as the E3-type small ubiquitin-like modifier ligase PIAS2 and the proto-oncogene FBI-1/Pokemon in human endothelial cells, that are regulated in a time dependent manner by Ang-(1-7), and are mainly part of metabolic pathways related to cell death and cell survival, whereby PIAS2 is regulated through posttranslational modification after short-term stimulation with Ang-(1-7).
Abstract 416: Inhibition of Metabolism of Endogenous AEA and 2-AG Reduces Ang II Induced Uterine Artery Contraction at Early Pregnancy in a Transgenic Model of Preeclampsia
Authors: Pulgar Victor M , Yamaleyeva Liliya M , Varagic Jasmina , Bader Michael , Dechend Ralf , Brosnihan K. Bridget
Published: March 2022

Recent evidence of interactions between the renin-angiotensin and the endocannabinoid systems suggests a regulatory role for endogenous cannabinoids (ECs) in modulating Ang II vascular responses. Endogenous levels of the ECs, AEA and 2-AG, are controlled by the hydrolytic enzymes FAAH and MAGL, respectively. We investigated the role of AEA and 2-AG as modulators of Ang II contraction in uterine arteries (UA) in a transgenic pregnant rat, in which the female expressing human angiotensinogen (AGT) is mated to the male with human renin (REN), producing a preeclamptic pregnancy. Uterine arteries were isolated from pregnant transgenic
h
AGTx
h
REN rats at 7 days of gestational age (n=4). Arterial segments were mounted in a wire myograph for determinations of isometric tension (DMT USA, 620M). Concentration response curves to Ang II (10
-11
-10
-8
M) were performed in control conditions and after preincubation with enzyme inhibitors: URB597 (FAAH) or JZL184 (MAGL) at 10
-6
M. Data were fitted to a dose response curve, maximal responses (Ang II
MAX
) were expressed as percent of maximal response to 75 mM KCl (% K
MAX
) and sensitivity as pD
2
(pD
2
= -Log [EC
50
]). Inhibition of FAAH attenuated both maximal response and sensitivity (Ang II
MAX
76±9 vs 105±3, p<0.05; pD
2
8.7±0.09 vs 9.1±0.04, p<0.05) and inhibition of MAGL lowered Ang II sensitivity (Ang II
MAX
83±15; pD
2
8.8±0.01, p<0.05),
Figure.
Thus, by inhibiting the enzymes involved in breakdown of ECs, we demonstrate a functional EC system in the UA that counteracts Ang II contraction. Whether the buffering capacity of the EC system is altered in this model of preeclampsia relative to normal pregnancy is under investigation.
Abstract 418: Angiotensin II Evokes Acute ATP and H
2
O
2
Release in the Kidney
Authors: Palygin Oleg , Levchenko Vladislav , Cowley Allen W , Staruschenko Alexander
Published: March 2022

The study of the functional processes in the whole kidney presents a tremendous experimental challenge due to the structural and functional heterogeneity of the kidney tissues and rapid transient changes in the concentrations of the signaling molecules. The purines, like adenosine-5’-triphosphate (ATP) as well as reactive oxygen species including H
2
O
2
are among the most important paracrine signaling agents. Angiotensin II (AngII) directly regulates many processes in the body and is strongly linked to the hypertension. We recently developed a novel approach based on the use of enzymatic biosensors suitable for real-time measurements of endogenous substances release in freshly isolated rat kidney. AngII-induced changes of interstitial concentration of ATP and H
2
O
2
were determined in Sprague Dawley (SD) and Dahl salt sensitive (SS) rats. Freshly isolated kidneys of the SD or SS rats were infused with AngII (1 μM) under constant laminar flow. The maximum amplitude of the AngII induced H
2
O
2
responses averaged 70 ± 15, 189 ± 58 and 301 ± 91 nM for SD and SS rats fed a low (0.4%) and high salt (4%; 3 weeks) diets, respectively. The H
2
O
2
response exhibited a slow increase to a stable maximal level that lasted during the rest of the perfusion time. The average production rate was 67 ± 17, 187 ± 55 and 294 ± 81 nM/s for SD, and SS rats fed low and high salt diets. The kinetics of ATP release had different characteristics compared to H
2
O
2
release with rapid increase in concentration triggered by AngII and followed by a slow decay of the signal due to ATP conversion to adenosine. The maximum amplitudes averaged 449 ± 145, 630 ± 49 and 1087 ± 96 nM for SD rats and SS rats fed low and high salts. The ATP production rates were 257 ± 100, 496 ± 202 and 829 ± 319 nM/s for the corresponding groups. To test AT receptor subtype mediated the acute release of ATP and H
2
O
2
in response to AngII, we used an AT
1
receptor antagonist losartan. Losartan (10 μM) inhibited the release of both ATP and H
2
O
2
in response to AngII application. Thus, these experiments reveal that established approach can be successfully used to analyze acute changes in endogenous substances in response to drug perfusion. Importantly, these data demonstrate that perfusion of the kidney with AngII rapidly induced release of both ATP and H
2
O
2
.
Abstract 42: A Novel Role of Proximal Tubular Na+/H+ Exchanger 3 (NHE3) in Angiotensin II-Induced Hypertension in Nhe3-/- Mice with Transgenic Rescue of NHE3 in Intestines
Authors: Li Xiao C , Shull Gary E , Miguel-Qin Elisa , Zhuo Jia L
Published: March 2022

The Na
+/
H
+
Exchanger 3 (NHE3) plays an important role in the regulation of Na
+
and HCO
3
-
reabsorption in proximal tubules of the kidney, but its contribution to the renal mechanisms of angiotensin II (ANG II)-induced hypertension remains unknown. Using global NHE3-deficient mice with transgenic rescue of the NHE3 gene in intestines (tg

Nhe3
-/-

) as a model of proximal tubule-specific

Nhe3
-/-

mice, we tested the hypothesis that deletion of NHE3 selectively in proximal tubules impairs long-term blood pressure responses to extracellular and intracellular ANG II. Three groups of wild-type (tg

Nhe3
+/+

) and six groups of tg

Nhe3
-/-

mice (n=>8 per group) were treated with vehicle, ANG II (40 ng/min, i.p.), or in vivo adenovirus-mediated transfer of an intracellular ANG II fusion protein, ECFP/ANG II, for 2 weeks. Under basal conditions, tgNhe3
-/-
mice had significantly lower systolic blood pressure (SBP) (tg

Nhe3
+/+

: 119±3 mmHg vs. tg

Nhe3
-/-

: 109±3 mmHg,
p
<0.01), glomerular filtration rate (tg

Nhe3
+/+

: 148.7±13.0 vs. tg

Nhe3
-/-

: 83.9±5.1 μl/min,
p
<0.01), 24 h urine excretion (tg

Nhe3
+/+

: 1.25±0.15 vs. tg

Nhe3
-/-

: 0.68±0.10 ml,
p
<0.05), 24 h urinary Na
+
excretion (tg

Nhe3
+/+

: 210.5.1±10.3 vs. tg

Nhe3
-/-

: 36.3±2.3 μmol,
p
<0.01), and 24 h urinary K
+
excretion (tg

Nhe3
+/+

: 343.8±19.4 vs. tg

Nhe3
-/-

: 125.5±13.3 μmol,
p
<0.01). By contrast, basal plasma ANG II (tgNhe3
+/+
: 303.4±26.9 vs. tg

Nhe3
-/-

: 397.7±27.2 pg/ml,
p
<0.05), aldosterone (tgNhe3
+/+
: 506±26.3 vs. tg

Nhe3
-/-

: 759.9±12.7 pg/ml,
p
<0.01), and the expression of Na
+
/HCO
3
-
cotransporter and Na
+
/K
+
-ATPase proteins in proximal tubules (
p
<0.01) were significantly increased in tg

Nhe3
-/-

mice. Deletion of NHE3 selectively in proximal tubules significantly attenuated the SBP responses to exogenous ANG II (tg

Nhe3
+/+

: 50±3 vs. tg

Nhe3
-/-

: 33±5 mmHg,
p
<0.01) and intracellular ECFP/ANG II (tg

Nhe3
+/+

: 18±3 vs. tg

Nhe3
-/-

: 6±2 mmHg,
p
<0.01) in tg

Nhe3
-/-

mice. 24 h urine and urinary Na
+
excretory responses to ANG II were also attenuated in these mice (
p
<0.01), whereas ECFP/ANG II had no further effects on these responses. We concluded that NHE3 in proximal tubules of the kidney is required for maintaining long-term blood pressure responses to extracellular and intracellular ANG II and body salt and fluid homeostasis.
Abstract 425: Beneficial Effects of BIA 5-1058 in a Genetic Model of Salt-sensitive Hypertension and Heart Failure
Authors: Igreja Bruno , Pires Nuno M , Wright Lyndon C , Soares-da-Silva Patrício
Published: March 2022

Hyperactivity of Sympathetic Nervous System (SNS) is implicated in numerous cardiovascular diseases. One strategy to slow down the SNS drive is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine-β-hydroxylase (DβH), the enzyme that catalyses the conversion of dopamine (DA) to NE in sympathetic nerves. BIA 5-1058 is a new DβH inhibitor that decreases NE and increases DA levels in peripheral sympathetically innervated tissues.

It was previously shown that BIA 5-1058 dose-dependently reduces blood pressure (BP) in spontaneously hypertensive rats. The Dahl salt-sensitive (Dahl/SS) rat is known to develop systemic hypertension, over 200 mmHg, due to combined pressure and volume overload, which results in compensated left ventricle (LV) hypertrophy after five to six weeks and cardiac failure with LV dilation and contractile dysfunction after 10 to 12 weeks of high-salt (HS) diet. The aim of the present study was to evaluate the effect of BIA 5-1058 in salt-induced hypertension and heart failure in the Dahl/SS rat.

To evaluate the effect of acute BIA 5-1058 treatment on hypertension, six Dahl/SS rats were implanted with telemetry device TA11PA-C40 (DSI) and fed a HS (8% NaCl) diet. Salt-induced hypertensive animals were used to evaluate two different doses (10 and 100 mg/Kg) of BIA 5-1058 on BP. Afterwards, a second cohort of rats were used to evaluate the effect of prolonged BIA 5-1058 treatment (30 mg/Kg/day) on the survival rate of Dahl/SS rats fed a HS diet.

The reduction in the sympathetic tone attained by DβH inhibition with BIA 5-1058 showed a dose- and time-dependent effect on systolic (SBP) and diastolic (DBP) blood pressure. The maximum reduction on SBP was -16.1 and -22.9 mmHg for 10 and 100 mg/Kg, respectively. Likewise, the maximum reduction on DBP was -18.8 and -23.3 mmHg for 10 and 100 mg/Kg, respectively. Survival rate of Dahl/SS rats fed a HS diet demonstrated that BIA 5-1058, beyond BP reduction, shows beneficial effects to the heart of Dahl/SS rats fed a HS diet with a significant 19-day increase in median survival.

In conclusion, the use of DβH inhibitors, like BIA 5-1058, is a promising approach to treat hypertension, heart failure and others cardiovascular diseases where a reduction in the sympathetic tone has beneficial effects.
Abstract 436: Administering Leptin into the Rostral Ventral Lateral Medulla (RVLM) Augments Blood Pressure and Renal Sympathetic Nerve Activity
Authors: Barnes Maria J , McDougal David M
Published: March 2022

Initially described as a hormone that regulates energy homeostasis, we now know that leptin is an integrative hormone that responds to and regulates various systems, in particular, the renal sympathetic nervous system. The rostral ventral lateral medulla (RVLM) contains neurons which evoke changes of arterial blood pressure by controlling the tonic and phasic activation of sympathetic activity. Thus, we sought to determine if leptin regulates blood pressure and renal sympathetic activity within the RVLM. Utilizing immunohistochemistry, we observed that neurons within the RVLM that receive sympathetic projections from the kidney express leptin receptors on them. We have also demonstrated that these neurons are responsive to low doses of leptin and evoke significant increases in blood pressure, renal sympathetic nerve activity and heart rate. Taken together, these data demonstrate that leptin can regulate blood pressure and renal sympathetic nerve activity within the RVLM.
Abstract 437: Inhibition of Endoplasmic Reticulum Function in Pre-sympathetic PVN Neurons by Thapsigargin Increased Neuronal Excitability and Sympathetic Nerve Activity
Authors: Chen Qing Hui , Gui Le , Chapp Andrew , Larson Robert , Huber Michael , Zhu Jianhua
Published: March 2022

Control of sympathetic outflow by pre-sympathetic paraventricular nucleus (PVN) neurons is partially mediated through a monosynaptic pathway that innervates the rostral ventrolateral medulla (RVLM). Here we investigated the role of the thapsigargin (TG), the endoplasmic reticulum (ER) Ca
2+
ATPase inhibitor, in regulating the in vitro excitability of PVN neurons projecting to RVLM (PVN-RVLM) under brain slice preparation. In current-clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22 ± 2 Hz (n=7) and was significantly greater (
P
< 0.05) in the presence of TG (0.5
μ
M) (32 ± 4 Hz, n=5). Next, the effect of Ca
2+
store depletion from the ER in regulating SNA and blood pressure was assessed by bilateral PVN microinjection of TG in anesthetized rats. PVN injection of TG (0.15, 0.3 0.75 and 1.5 nmol/100nl) increased the renal sympathetic nerve activity (SNA) and mean arterial pressure (MAP) in a dose-dependent manner. Maximum increase in renal SNA and MAP elicited by PVN TG (0.75 nmol/100nl; n=3) were 105 ± 31% and 18 ± 11 mmHg respectively. Pre-treatment of CyPPA (5 nmol/100 nl), a SK channel activator/opener (n=3), remarkably attenuated the PVN TG evoked sympathoexcitatory and pressor responses (7 ± 7% for renal SNA and 4 ± 7 mmHg for MAP). PVN injection of CyPPA did not significantly alter the baseline of renal SNA and MAP. Our data indicate that inhibition of ER function by TG contributes to the enhanced
in vitro
excitability of PVN-RVLM neurons and increased
in vivo
renal SNA and MAP. The effect of TG on the excitability of pre-sympathetic PVN neurons and sympathetic outflow through the Ca
2+
store depletion from the ER may involve the down-regulation of SK channel function. Support: NSDP2010008 (JZ) and AHA10SDG2640130 (CQH).
Abstract 440: Activation of the Brain Renin-angiotensin System is Critically Involved in the Hypertension Associated With Bbs
Authors: Guo Deng-Fu , Morgan Donald A , Sheffield Val C , Rahmouni Kamal
Published: March 2022

Bardet-Biedl syndrome (BBS) is a human autosomal recessive disorder characterized by a pleiotropic of phenotypes including obesity, renal abnormalities, mental retardation, retinal pigmentary dystrophy, polydactyly and hypogenitalism. Hypertension is also common in BBS patients, except BBS2. Consistent with this, we previously demonstrated that obese Bbs2 knockout mice are normotensive while obese Bbs4 null mice are hypertensive. However, the underlying molecular mechanisms of hypertension in BBS remain poorly defined. Using BBS mice, we tested whether abnormalities in the brain renin-angiotensin system (RAS) may contribute to the increased arterial pressure in BBS. Using real time PCR, we found that mRNA expression of RAS components were differentially altered in Bbs2
-/-
and Bbs4
-/-
mice. Compared to wild type controls, the hypertensive Bbs4
-/-
mice exhibited significantly (P<0.05) elevated expression of renin (160%), angiotensin converting enzyme (ACE, 185%), angiotensin type 1a receptor (AT1aR, 143%) and AT1bR (244%) in the brain. Notably, while the expression of ACE was increased (154%) in the brain of the normotensive Bbs2
-/-
mice, the expression of renin, AT1aR and AT1bR were significantly (P<0.05) decreased by 56%, 60% and 36%, respectively, relative to controls. Next, we examined the functional consequences of the elevated brain RAS components in Bbs4
-/-
mice. Consistent with our previous findings, radiotelemetric mean arterial pressure was significantly (P<0.05) elevated in the obese Bbs4
-/-
mice (114±3 mmHg) relative to wild type controls (101±1 mmHg). Moreover, Bbs4
-/-
mice exhibited an exaggerated decrease in arterial pressure in response to intracerebroventricular (ICV) administration of losartan (AT1 receptor antagonist). Indeed, ICV losartan (10 g) decreased mean arterial pressure by 24±7 mmHg in Bbs4
-/-
mice vs. 5±10 mmHg in wild type controls (P<0.05). Our data indicate that overactivity of the brain RAS may account for the high blood pressure associated with BBS. This study provides a novel molecular mechanism for the hypertension associated with BBS.
Abstract 469: The Role of PP2A in Angiotensin AT2R and Dopamine-1 Receptor Membrane Recruitment in Human Renal Proximal Tubule Cells
Authors: Opper Phuong B , Gildea John J , Felder Robin A
Published: March 2022

The renal dopamine-1 receptor (D1R) and angiotensin type 2 receptor (AT2R) are two important receptors that regulate renal sodium reabsorption through heterologous and autologous plasma membrane recruitment following stimulation of protein kinase A (PKA) via D1R and protein kinase G (PKG) via AT2R. Using the cell permeable analog of PKA (8Br-cAMP) along with the PKG analog 8Br-cGMP, we did not find additive effects, which led to our hypothesis that downstream protein phosphatase 2A (PP2A) is the common link in the D1R/AT2R interaction. To test our hypothesis, we used two human renal proximal tubule cell lines (RPTC): one with normal D1R coupling (nRPTC) to adenylyl cyclase (AC) and a 2nd cell line with a defect in D1R coupling to AC (uncoupled or uRPTC). A dose response curve for 8Br-cAMP and 8Br-cGMP using cell surface Dylight-649-AngIII binding for 1 hr in the presence of losartan (a measure of surface AT2R) showed maximal recruitment at 1 mM for both 8Br-cAMP and 8Br-cGMP. EC50 of 8Br-cAMP was 129.9±60.58 μM (log EC50 was -3.9). EC50 of 8Br-cGMP was 99.17±48.13 μM (log EC50 was -4.0) A 1 hr addition of 1 mM 8Br-cAMP or 8Br-cGMP resulted in increased PP2A activity. Specifically, PP2A activity increased by 1.59±0.24 fold (N=3, P<0.05 vs vehicle (VEH, baseline value of 1216 pmoles of phosphate at 650 nm) when 8Br-cAMP was added. The addition of 8Br-cGMP induced a 1.48±0.33 fold increase in PP2A (N=3, P< 0.05 vs VEH at 1216 pmoles of phosphate at 650 nm). Using flow cytometry for cell surface D1R and AT2R, recruitment of either receptor (AT2R or D1R) was completely blocked by okadaic acid (OA, a specific PP2A phosphatase inhibitor at 10 nM), whether stimulating D1R with fenoldopam (FEN), AT2R with AngIII, or using cell permeable downstream second messenger agonists 8Br-cAMP or 8Br-cGMP. Similarly, addition of the AC inhibitor SQ 22536 (10 μM) or the guanylyl cyclase (GC) inhibitor ODQ (10 μM) completely blocked FEN, AngIII, 8Br-cAMP and 8Br-cGMP-induced D1R and AT2R cell surface recruitment. In summary, we determined that PP2A is the common mediator of D1R and AT2R cell surface recruitment and may be an important target for regulating renal sodium homeostasis.
Abstract 491: Nox4 Inhibition, by GKT 137831, Decreases Obesity-Associated Vascular Dysfunction and Pro-Fibrotic Effects
Authors: Montezano Augusto C , Nguyen Dinh Cat Aurelie , Even Sarah E , Antunes Tayze , Briet Marie , He Ying , Callera Glaucia E , McCulloch Jim , Carswell Andrew , Jandeleit-Dahm Karin , Cooper Mark E , Szyndralewiez Cedric , Page Patrick , Touyz Rhian M
Published: March 2022

The pathophysiological role of Nox4 remains elusive and controversial. Even though Nox4 expression is increased in hypertension, diabetes and obesity, some studies demonstrate that Nox4 deficiency predisposes to obesity and insulin resistance. We showed that Nox1/4 inhibition is renoprotective in diabetes. Whether Nox4 influences vascular function and kidney status in obesity associated with mild diabetes is unclear. We hypothesized that Nox4-specific inhibition is protective against obesity-associated vascular and kidney damage. We studied db/m (control) and db/db mice (obese) mice for 16 weeks receiving 1) vehicle; 2) low dose Nox4 inhibitor, GKT137831 (GKT) (20 mg) and 3) high dose GKT (60 mg) for 16 weeks. Body weight increased in db/db (61.8g ± 0.95) versus controls (33.5g ± 0.72, p<0.05). Plasma glucose and albuminuria were slightly increased in db/db. GKT did not influence body weight but reduced epididymal fat mass in db/db (20% increase, p<0.05). Blood pressure was similar in all groups. Kidney weight (25%) and markers of renal fibrosis, such as, fibronectin (60%), pro-collagen I (PCI - 50%) and TGFβ (30%), were increased in db/db versus controls (p<0.05). High dose GKT decreased renal expression of fibronectin and PCI in db/db. Renal ERK1/2 activation was ameliorated by high dose GKT in db/db (vehicle, 65% vs GKT, 2.3%, p<0.05). Plasma 8-isoprostanes (db/db: 1263±96 vs db/m: 936±35), marker of systemic oxidative stress, and kidney hydrogen peroxide (db/db: 1 fold increase vs db/m) levels were increased in db/db, effects blocked by GKT. Endothelial dysfunction in db/db mice was not affected by GKT, but decreased NE-induced vascular contraction in db/db (pD2: db/db vs. db/db high dose, 6.4±0.1 vs. 5.6 ± 0.1, p<0.001). Reduced contraction by GKT was associated with decreased Rho kinase activity (db/db: 84% increase vs db/db+GKT 60 mg: 29% increase, p<0.05). Our findings suggest a role for Nox4 in obesity-associated kidney and vascular damage/dysfunction, important factors predisposing to cardiovascular disease in obesity. Mechanisms underlying Nox4 effects involve oxidative stress and Rho kinase.