bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 381: Placental Growth Factor Controls Splenic Immunity to Drive Hypertension
Authors: Carnevale Daniela , Pallante Fabio , Fardella Valentina , Iacobucci Roberta , Fardella Stefania , De Lucia Massimiliano , Lembo Giuseppe
Published: March 2022

Although several therapeutical strategies have been developed against the main components involved in the regulation of blood pressure (BP), an optimum control in some hypertensive patients is still flawed, suggesting that mechanisms still unidentified, sustain the increase of BP. Interestingly, an emerging area of investigation revealed that adaptive immunity is a central player of hypertension, since mice devoid of T lymphocytes were protected from AngII-induced hypertension. However, how hypertensive stimuli, like AngII, can afford this, still represents a fascinating enigma, challenging the search for molecular players, capable to bridge vascular responses to immune cells and probably orchestrating the still unveiled role of immune system in hypertension.

Here we demonstrate an unprecedented splenic immune pathway driven by Placental Growth Factor (PlGF), an “angiogenic cytokine” acting as a bridge between the cardiovascular and immune system during onset of hypertension. We found that the typical hypertensive response induced by chronic AngII infusion, was completely prevented in mice with genetic deletion of PlGF, as well as the typical end organ damage induced by hypertension and immune cells infiltration in target organs. To determine whether the involvement of PlGF in immune system activation and T cells infiltration during AngII-induced hypertension had a causal role or was merely a consequence of the failure in BP raise, we analyzed both vessels and kidney, early after AngII infusion, i.e. before BP increase. Strikingly, PlGF absence protected from early infiltration of both CD4+ and CD8+ T cells. More interesting, we unveil that the spleen is indispensable in the genesis of hypertension by releasing PlGF, that orchestrates the T cell response, necessary for blood pressure raising. In particular, we show that splenic PlGF acts as a negative checkpoint of a tissue protein of inflammation (Timp3), a repressor of T cell costimulation axis, thus allowing T cell activation and egression from spleen. Basically, this breakthrough in a key immune pathway determining blood pressure elevation, holds a great translational promise for patients with refractory disease and multiple complications.
Abstract 382: Increasing Parasympathetic Function Improves the Autonomic Balance and Attenuates the Inflammatory Process During the Development of Hypertension in Spontaneously Hypertensive Rats
Authors: Lataro Renata Maria , Silva Carlos A , Fazan Rubens , Salgado Helio C
Published: March 2022

Autonomic dysfunction and inflammation contribute to the pathophysiology of hypertension. Therefore, an increase in parasympathetic function may have benefic effects during the development of arterial hypertension. We investigated the chronic effect (16 weeks) of the acetylcholinesterase inhibitor donepezil on arterial pressure (AP), heart rate (HR), vagal tone, AP and pulse interval (PI) variability, plasma noradrenaline and pro-inflammatory plasma cytokines in spontaneously hypertensive rats (SHR). DON administration (minipumps, 2 mg/kg/day, n=8) started with SHR aging 5 weeks. Control SHR (n=7) were implanted with empty minipumps. At the end of 16 weeks, femoral AP was recorded in conscious state and methyl atropine provided the evaluation of the vagal tone. Plasma cytokines were analyzed by ELISA. DON promoted: decrease in systolic AP (178±8 vs 202±5 mmHg in control) and HR (304±9 vs 354±13 bpm in control); increase in vagal tone (105±11 vs 64±5 bpm in control); no change of plasma noradrenaline (8.7±0.6 vs 9.1±1.1ng/mL in control); reduction of AP variance (39±7 vs 63±5 mmHg
2
in control), power of the low frequency band (LF) of the systolic AP spectrum (4±1 vs 12±3 mmHg
2
in control) and LF/HF ratio of PI spectrum (0.2±0.03 vs 0.5±0.005 in control). DON reduced the pro-inflammatory cytokines (pg/mL): TNF-α (22±9 vs 54±5 in control), IFN-γ (11±5 vs 27±5 in control) and IL-6 (22±4 vs 38±5 in control). In conclusion, the increase in parasympathetic function by means of the chronic blockade of acetylcholinesterase with DON improved the cardiovascular autonomic balance and attenuated the inflammatory process during the development of hypertension in SHR.
Abstract 387: Role of STAT3 in Collagen Deposition and Organization in the Normal and Hypertensive Heart
Authors: Zouein Fouad A , Kurdi Mazen , Booz George W , Fuseler John
Published: March 2022

Hypertension causes concentric hypertrophy of the left ventricle that may progress to systolic heart failure by means not defined. We reported that mice with a S727A reduced function mutation in STAT3 have reduced systolic function with angiotensin II (Ang II)-induced hypertension and foci of fibrotic collagen in the myocardium as occurs in hypertensive heart disease. We applied image analysis of relative density (IOD/A) and fractal dimension (D) to quantify foci. Hypertension in WT mice did not affect % area and relative density, but D values decreased (P<0.001) suggesting tighter packing of fibrotic collagen stiffening the myocardium. SA/SA+saline hearts showed significant increases in fibrotic collagen (P=0.025) and density (P<0.001) vs. WT hearts. D values indicate an increase (P=0.002) in chaos of collagen matrix. In SA/SA mice Ang II caused remodeling of myocardial collagen with loss of myocytes within fibrotic foci. Hypertension in SA/SA hearts induced further increase (P<0.001) in collagen, while the relative density was significantly less (P=0.015) than the SA/SA+saline group but greater than WT+saline and WT+Ang II groups. The decrease in collagen density was associated with a large (P<0.001) increase in D values, indicating collagen in fibrotic foci was highly disorganized and more space filling. Loss of myocytes and their replacement by chaotic and randomly organized collagen may further weaken the myocardium leading to systolic heart failure. Our results show that STAT3 is essential in normal regulation and organization of the collagen matrix in the heart. Suppression of STAT3 results in fibrotic lesions in the myocardium that are exacerbated by hypertension.
Abstract 396: Enhanced VEGF and ETS-1 Recruitment by T Reg- Heme Oxygenase- 1 Module Increases Blood Flow in Post-infarction Myocardium in SCID Mice
Authors: Touchon Robert , Thompson Ellen , Sodhi Komal , Favero Gaia , Rezzani Rita , Fabrizio Rodella Luigi , Abraham Nader G , Shapiro Joseph , Dial Larry
Published: March 2022

Introduction:
Post-infarction cardiomyopathy has been ascribed to chronic inflammation, increased oxidative stress and insufficient angiogenesis. T lymphocyte mediated immune response plays an important role in post-infarct cardiac remodeling. T regulatory lymphocytes (Treg) are a subpopulation of T helper lymphocytes with a regulatory and immunosuppressive role. Heme oxygenase (HO)-1 induction improves heart function after ischemic damage by its anti-oxidative effect. To assess the role of the HO-1 mediated increased levels of T-reg lymphocytes in immunosuppressed mice in a model of post-infarcted heart failure.

Methods:
We compared the effect of HO-1 induction on post-infarction myocardium induced by left anterior coronary artery ligation in T-lymphocyte immunosuppressed mice (BALB SCID)
.
Mice were divided into 4 groups: sham, myocardial infarction (MI), MI treated with the HO-1 inducer cobalt protoporphyrin (CoPP) with and without the HO activity inhibitor stannous mesoporphyrin (SnMP). Thirty days after surgery all mice underwent echocardiography, immunohistochemistry and an assessment of T-lymphocyte subpopulations (Treg cells) via fluorescence activated cell sorting (FACS).

Results:
Mice with MI had increased levels of inflammatory cytokines (p<0.05) and lower capillary density (SCID: 1.2±0.7% vs control: 4.3±0.9%; p<0.01). Left ventricle end diastolic area (EDA) was reduced in CoPP treated compared to the MI group (EDA: MI: 0.22 ±0.02 cm; MI+CoPP: 0.17±0.03 cm; p<0.01). In CoPP treated SCID mice the T-reg subpopulation measured by FACS analysis (20±3% vs 5±3%; p<0.01) and immunohistochemistry (p<0.02) was significantly increased as compared to the MI group. Increased expression of the angiogenic markers VEGF, FGF and ETS-1 (p<0.02) was found in CoPP treated animals as compared to MI animals. These beneficial effects were reversed by SnMP.

Conclusion:
Upregulation of HO-1 increases T regulatory cells, reverses dysfunctional remodeling and enhances angiogenesis and peri-infarct survival in the post-infarction myocardium in immunosuppressed mice. Targeted enhancement of T-reg cells via HO-1 induction suggests adjuvant avenues for reducing morbidity and mortality in patients with post-MI heart failure.
Abstract 398: Mice Containing the Human Gilbert’S Syndrome Mutation (UGT1A1*28) Are Resistant to the Development of Type II Diabetes and Deposition of Body Fat on a High Fat Diet
Authors: Hosick Peter A , Stec David E
Published: March 2022

Heme oxygenase-1 (HO-1) induction has been previously demonstrated to reduce body weight and prevent type II diabetes in obese mice. However, the role of increased bilirubin production in this response is unknown. Gilbert’s syndrome is derived from a mutation in the hepatic UGT1A1 gene which results in decreased conjugation and increased levels of plasma biliribin independent of changes in HO-1. In order to determine the role of increased plasma bilirubin on the development of type II diabetes and obesity, we placed mice whose only functioning UGT1A1 gene contained the human Gilbert’s syndrome mutation (UGT1A1*28, n=4) as well as C57BL/6J control mice (n=5) on a high fat (60%) diet for 35 weeks. Plasma bilirubin levels were significantly increased in the UGT*28 mice as compared to controls and averaged 1.65 ± 0.2 vs. 1.18 ± 0.1 mg/dL (P<0.05). UGT*28 mice exhibited significantly decreased body weights (14-17%, P<0.05) early (4-8 weeks) and late (29-32) after high fat feeding as compared to control mice. Fat mass as measured by Echo-MRI was significantly decreased by 15-20% on weeks 18,24,30,35 as compared to control mice (P<0.05). Lean mass was significantly increased by 7-10% in UGT*28 mice as compared to control mice at 18, 24, and 35 weeks (P<0.05). Fasting blood glucose levels in UGT*28 mice over the entire study were significantly lower as compared to control and averaged 94 ± 3 vs. 138 ± 9 mg/dL (P<0.05). Plasma insulin levels were also significantly decreased in UGT*28 mice over the study averaging 1.8 ± 0.1 vs. 2.9 ± 0.2 ng/mL (P<0.05). No differences in food intake or oxygen consumption were observed in UGT*28 mice as compared to control mice. These results suggest that moderate increases in plasma bilirubin can attenuate the increase in fat mass accumulation and normalize insulin sensitivity in response to a high fat diet.
Abstract 40: cGMP-induced Inhibition of NKCC2 is Blunted in Thick Ascending Limbs from Dahl Salt Sensitive Rats: Role of Decreased PDE2
Authors: Haque Mohammed Z , Ares Gustavo R , Ortiz Pablo A
Published: March 2022

The natriuretic effect NO is decreased in the Dahl Salt-Sensitive (SS) rat. This is in part due to decreased NO-induced inhibition of thick ascending limb (TAL) NaCl reabsorption. However, the mechanism responsible for this defect in SS rats is unclear. In normal rats, NO inhibits TAL NaCl reabsorption via cGMP. cGMP stimulates phosphodiesterase 2 (PDE2) which degrades cAMP and in turn decreases apical surface levels of the Na-K-2Cl cotransporter NKCC2. We hypothesized that the blunted effect of NO in TALs from SS rats is in part due to a decreased response to cGMP and reduced PDE2 expression. To test our hypothesis, we isolated TALs from adult SS and Dahl Salt-Resistant (SR) rats fed a normal salt diet and measured the effect of cGMP on apical NKCC2 levels by surface biotinylation and Western blot. Incubation of TALs (20 min) with the membrane permeable cGMP analogue db-cGMP (250 μM) decreased surface NKCC2 in SR rats by 22 ± 6% (
p
<0.05), while 500 μM decreased it to a maximum of 40 ± 6% (
p
<0.01). In SS TALs, the effect db-cGMP was blunted (
p
<0.05 vs SR) and only tended to decrease surface NKCC2 (-10± 9%) at 500 μM. Consistent with a decreased response to cGMP, the expression of PDE2 was 20 ± 5% lower in TALs from SS rats (
p
<0.05). Finally, we tested whether the effect of the PDE2 selective agonist DM-cBIMP was decreased in SS rats. Incubation of SR TALs with DM-cBIMP decreased surface NKCC2 expression in a dose-dependent manner with a maximum inhibition of 41 ± 6% at 250 μM. In SS TALs, the response was significantly blunted with a maximal inhibition of 20 ± 6% at 250 μM (
p
<0.05 vs. SR). We concluded that the decreased response to cGMP in TALs from SS rats is in part due to decreased PDE2 activation and expression. This mechanism is likely responsible for the blunted inhibitory effect of NO in TAL NaCl reabsorption in SS rats and may be involved in the development of salt-sensitive hypertension.
Abstract 411: Arterial Stiffening Precedes Systolic Hypertension in Diet-induced Obese Mice and Is Reversed by Weight Loss
Authors: Seta Francesca , Weisbrod Robert M , Shiang Tina , Al Sayah Leona , Fry Jessica L , Bajpai Saumendra , Reinhart-King Cynthia A , Lob Heinrich E , Santhanam Lakshmi , Mitchell Gary , Cohen Richard A
Published: March 2022

BACKGROUND.
Aortic stiffness (AS) is an independent predictor of cardiovascular events and is associated with hypertension (HTN) in aging and obesity however it is unclear whether HTN causes AS or AS could develop in advance of HTN. C57Bl/6J mice fed a diet rich in fat and sucrose (HFHS) develop obesity mimicking the metabolic syndrome.

AIMS.
We sought to determine: 1) whether AS occurs in HFHS-fed mice; 2) the relation between AS and HTN in such a model and 2) whether weight loss could improve AS and HTN in obese mice.

METHODS.
Pulse wave velocity (PWV) was measured by ultrasound as an in vivo index of arterial stiffness and confirmed invasively with high-fidelity pressure catheters. Blood pressure was measured by radiotelemetry in conscious mice. Weight loss was achieved by reversing mice to ND after 5 months of HFHS with bi-weekly assessments of metabolic and cardiovascular parameters.

RESULTS.
AS developed in HFHS-fed mice within 2 months (PWV from 2.3 ± 0.2 to 4.1 ± 0.5 m/s, n=6, mean ± SEM, p<0.05) and remained elevated up to 8 months (5.1 ± 0.5 m/s, n=10). Systolic and mean arterial pressures were significantly elevated after 6 months of HFHS (SBP: from 119.2 ± 1.8 to 140.4 ± 2.5 mmHg and MAP: from 106.9 ± 1.9 to 123 ± 1.6 mmHg; n=6). Within two months, when AS was fully developed, aortas from HFHS-fed mice had impaired endothelium-dependent relaxation and increased transglutaminase-2 activity, both in vitro indices of reduced NO bioactivity and ∼3-fold upregulation of pro-inflammatory cytokines TNFα, MCP-1 and MIP1α. Diet reversal induced a return to normal weight within 8 weeks accompanied by a 2-fold reduction in fat mass and hyperinsulinemia. PWV was significantly reduced from 5.3 ± 0.7 to 3.4 ± 0.4 m/s after reversal to ND and SBP and MAP were decreased to normal values (from 138.1 ± 3.7 to 124.2 ± 2.7 mmHg and from 121.2 ± 3.9 to 109.5 ± 3.4 mmHg, respectively). Atomic force measurements on aortic rings, an in vitro indicator of structural stiffness of the intima, was increased after HFHS (52 ± 4.8, n=7 vs 24 ± 2.8 kPa in ND, n=10) and significantly decreased after reversal to ND (30 ± 3.7 kPa, n=8).

CONCLUSIONS.
In a model of dietary obesity, arterial stiffness precedes the onset of hypertension and is reversed by normalization of the metabolic state achieved by weight loss.
Abstract 419: Investigating the Role of Epidermal Growth Factor Receptors in Angiotensin II-induced Hypertrophic Signalling in Cardiomyocytes
Authors: Wang Zhen , Chan Hsiu-Wen , Chen Chen , Thomas Walter G , Paravicini Tamara M
Published: March 2022

Cardiac hypertrophy is characterized by stimulation of hypertrophic signalling pathways representing the reactivation of the ‘foetal gene program’ (hypertrophic genes) and activation of the MAPK pathway. Angiotensin II (AngII) stimulates cardiac hypertrophy, a process which may involve the transactivation of epidermal growth factor receptors (ErbBs). Three subtypes of ErbBs are present in the postnatal heart (ErbB1, 2, 4), yet their precise roles are poorly understood. We have shown that the ErbB4-selective ligand neuregulin 1 beta-1 (NRG1-β1) potently stimulates cardiomyocyte hypertrophy. We now aim to determine if AngII activates hypertrophic signalling in cardiomyocytes by transactivation of ErbBs, particularly ErbB4.

Ventricular cardiomyocytes from neonatal rats were treated with AngII (100 nM) or NRG1-β1 (10 nM) to induce hypertrophic signalling. ErbB1, 2, 4 were downregulated using shRNA and siRNA. Reporter assays were used to measure promoter activity of the hypertrophic genes myosin light chain 2v (MLC-2V), atrial natriuretic peptide (ANP) and cyclin D. ERK1/2 activation was measured via Western blot.

NRG caused robust activation of the hypertrophic gene MLC-2V (11.3 fold increase versus untreated control) that was significantly reduced to 4.3-fold by silencing of ErbB4 (n = 5, P < 0.01), but not ErbB1 or ErbB2, confirming that ErbB4 activation contributes to hypertrophic signalling. However, knockdown of ErbB1, 2 or 4 did not affect AngII-induced activation of MLC-2V (5.8 fold increase versus untreated control). Similar results were seen for ANP and cyclin D. Similarly, ErbB4 knockdown reduced NRG1-β1-induced MAPK activation from 21.0-fold to 13.5 fold (n=4, p<0.05), whilst Ang II-induced MAPK activation was unaffected.

Cardiomyocyte hypertrophy can be promoted by activation of AT
1
and ErbB4 receptors. AngII-induced hypertrophic signalling (measured by promoter activity of MLC-2V, ANP and cyclin D and MAPK activation) may not involve transactivation of ErbB receptors. We are now extending these findings
in vivo;
we have established a model of cardiac-specific ErbB4 deletion using Cre/Lox recombination, and are undertaking echocardiographic analysis to measure cardiac hypertrophy and function.
Abstract 421: Dehydration Upregulates the Full-length (Pro)renin Receptor Expression in the Kidney of Wistar-Kyoto Rats and Spontaneously Hypertensive Rats
Authors: Tamura Yuma , Ito Osamu , Mori Nobuyoshi , Rong Rong , Takahashi Kazuhiro , Totsune Kazuhito , Kohzuki Masahiro
Published: March 2022

(Pro)renin receptor ((P)RR) was newly identified as a member of the renin-angiotensin system (RAS). (P)RR exists in two forms; full-length form (f(P)RR) and soluble form (s(P)RR). The truncated hydrophilic s(P)RR is generated by furin cleavage in the trans-Golgi network and is secreted into the extracellular space. Compared with Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR) demonstrated low urine concentration ability and resistance to vasopressin in a state of dehydration (DH). RAS system contributes to the regulation of body fluid balance, but roles of (P)RR in a state of dehydration (DH) are not clarified. The purpose of this study was to examine the effects of DH on the (P)RR expression in the kidney of WKY and SHR. Twelve week-old, male WKY and SHR were randomly divided into two groups (n=5 in each group); control and DH groups. DH was induced by water restriction for 72 hours in metabolic cage. After 72 hours, rats were killed, and (P)RR protein levels in plasma, urine and kidney were examined by using Western blot. (P)RR mRNA levels were examined by using competitive RT-PCR methods. In addition, furin protein levels were also investigated. DH increased urine osmolarity in WKY and SHR (4090±451 and 3796±429 mOsm/kg). DH significantly decreased the s(P)RR levels in the plasma of WKY and SHR (0.3- and 0.7-fold, P<0.01), but significantly increased in the urine of these rats (1.5- and 3.3-fold, P<0.05). DH significantly increased the f(P)RR levels in the renal homogenates, microsome fraction, and cytosol fraction of WKY and SHR (WKY: 2.3-, 3.0-, and 22.9-fold, SHR: 2.3-, 2.1-, and 7.9-fold, P<0.01). DH did not change the s(P)RR levels in the renal homogenates and cytosol fraction of these rats. In the kidney, DH did not change (P)RR mRNA levels in WKY, but significantly increased them in SHR (2.7-fold, P<0.05). DH significantly decreased furin protein levels in WKY (0.7-fold, P<0.05), but did not change them in SHR. These results indicate that DH upregulates the f(P)RR expression in the kidney of WKY and SHR. The DH-upregulated f(P)RR expression might be mediated through the decrease of furin levels in WKY and increase of (P)RR mRNA levels in SHR.
Abstract 422: Extracellular Domain of the (Pro)renin Receptor Participates in Dimerization
Authors: Suzuki-Nakagawa Chiharu , Nishimura Misa , Tsukamoto Tomoko , Aoyama Sho , Ebihara Akio , Suzuki Fumiaki , Nakagawa Tsutomu
Published: March 2022

The (pro)renin receptor [(P)RR] is a single transmembrane protein with a large N-terminal extracellular domain and a short C-terminal cytoplasmic domain. Three molecular forms of (P)RR are known: a full-length form, a C-terminal truncated soluble form [s(P)RR], and an N-terminal truncated transmembrane form (M8-9 fragment). It has been reported that (P)RR forms homodimer, but the region participating in dimerization is unknown. Here we report that the extracellular domain of (P)RR participate in dimerization. Using secretory glutathione S-transferase (GST) as a fusion tag, we developed an
in vivo
pull-down assay to investigate (P)RR dimerization in the cell. GST-tagged extracellular domain (amino acid residues 17-304) of human (P)RR [GST-ECD], as well as full-length (P)RR [GST-Full] and M8-9 fragment [GST-M8-9], was retained in the cell, while GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Consistent with this observation, immunofluorescence microscopy showed prominent localization of GST-ECD to the ER.
In vivo
GST pull-down experiments revealed that GST-ECD as well as GST-Full bound FLAG-tagged full-length (P)RR, whereas GST-M8-9 bound it little, if any, when transiently coexpressed in HEK293T cells. To investigate whether s(P)RR can form heterodimer with full-length (P)RR, we established a CHO cell line stably expressing human (P)RR with C-terminal 10His-tag. All three forms of (P)RR were detected in the cell lysate and only s(P)RR was detected in the culture medium. Full-length (P)RR and s(P)RR were prominently localized to the ER as observed by immunofluorescence microscopy. Pull down experiment using His-tag affinity resin showed coprecipitation of s(P)RR with full-length (P)RR. These results indicate that the extracellular domain of (P)RR participates in dimerization.
Abstract 427: Comparison of RF Renal Denervation Systems: Multiple Bipolar Electrodes With Blood Flow Control vs. Single Monopolar Electrode
Authors: Tunstall Radhika R , Schauer Travis , Cao Hong , Willard Martin
Published: March 2022

Introduction:
Boston Scientific’s Vessix
TM
Over-the-Wire (OTW) balloon catheter uses bipolar radiofrequency (RF) energy and blood flow control to deliver significantly less energy than monopolar RF renal denervation systems. We hypothesize that the greater energy used in monopolar RF systems increases the risk for non-target tissue injury and inconsistent lumenal lesion formation.

Methods:
A computational model was used to simulate the clinical settings and control algorithms of a monopolar single point catheter and bipolar balloon catheter. The electrical field was solved to calculate the Joule heating in tissue and a transient thermal model was solved for the temperature change for the duration of RF energy delivery.

In a swine model, RF treated renal arteries and select psoas muscles were rinsed and submerged in a beaker containing TTC (Triphenyl Tetrazolium Chloride) solution to aide in identification of thermal lesions. Non-viable tissue does not metabolize the stain and remains white, in contrast to red stained viable tissue.

Results:
The computational model predicted that the monopolar RF lesion varied due to electrode-tissue contact conditions. Lesion depth was dependent on the electrical conductivity of the artery wall and structures near the electrode. In contrast, the Vessix bipolar balloon produced consistent lesion pattern and depth due to fixed balloon apposition and blood flow control, focal energy delivery, and the temperature control algorithm.

TTC stained renal arteries treated with the bipolar balloon device had accurate & consistent electrode placement. The single electrode monopolar device had inconsistent tip placement, with lesion depths decreasing with decreasing treatment time. Non-target lesions (psoas muscle and bowel) were observed with the monopolar device. Psoas muscle thermal lesion depth decreased from 1.5mm with 120s ablation time (67% incidence) to 0.3mm with 30s ablation time (17% incidence) with a single electrode unipolar device (N=16). Non-target lesions were not observed with the bipolar balloon device (N=13).

Conclusions:
The BSC bipolar balloon renal denervation system had increased accuracy of electrode placement in the renal artery with no observations of non-target thermal injury.
Abstract 428: Epicardial Application of Resiniferatoxin During Coronary Ligation Prevents Cardiac Remodeling and Improves Cardiovascular Dysfunction in Rats With Heart Failure
Authors: Wang Hanjun , Wang Wei , Cornish Kurtis G. , Zucker Irving H.
Published: March 2022

Sympatho-excitation is a hallmark of the chronic heart failure (CHF) state and is positively related to the morbidity and prognosis of patients with CHF. Previous studies from this laboratory have shown that the enhanced cardiac sympathetic afferent reflex (CSAR), a sympatho-excitatory reflex originating in the heart, contributes to the elevated sympathetic tone in CHF. Therefore, a therapeutic strategy for abolishing the contribution of the CSAR to this sympatho-excitation may be beneficial in CHF patients. Here, we applied Resiniferatoxin (RTX, 50 μg/ml), an ultra-potent agonist of transient receptor potential vanilloid 1 (TRPV1), to the surface of the rat heart during myocardium infarction (MI) surgery in order to selectively delete the TRPV1 receptor from contributing to activation of the CSAR. This procedure largely abolished the enhanced CSAR in CHF rats (blood pressure change in response to cardiac application of capsaicin and bradykinin (10 μg/ml): capsaicin, 33±2 vs. 5±1 mmHg; bradykinin, 37±3 vs. 11±2 mmHg; CHF+Vehicle vs. CHF+RTX 9-11 weeks after MI). Compared to CHF+Vehicle rats (n=7), there was significantly decreased sympathetic tone (50±6 vs. 22±2 % of Max, P<0.05) and improved baroreflex sensitivity (gain: 2.1±0.1 vs. 3.1±0.3, P<0.05) in CHF+RTX rats (n=9). Importantly, we found that compared to CHF+ Vehicle rats, CHF+RTX rats had normal left ventricular end diastolic pressure (LVEDP: 24±2 vs. 6±1 mmHg, P<0.05) even with similar infarcted size (39±5 vs. 32±2 %) and similar ejection fraction (36±4% vs. 39±2%). Cardiac hypertrophy and lung edma in CHF rats were prevented by RTX treatment. The latter was highly consistent with lowered LVEDP in CHF+RTX rats. Molecular evidence showed that RTX treatment significantly attenuated the left ventricular expressions of the fibrosis marker, smooth muscle actin alpha/GAPDH: (0.77±0.02 vs. 0.55±0.02, n=6, P<0.05). Given that reduced cardiac fibrosis may increase cardiac compliance, this molecular evidence may provide a potential explanation for the lower LVEDP in CHF+RTX rats. In summary, these are the first data to our knowledge to show that selective cardiac sympathetic deafferentation may be a potential clinical therapy for improving cardiac and autonomic function in the CHF state.
Abstract 431: 24-Hour Imputed Aortic-to-Brachial Pulse Pressure Amplification is Associated with Left Ventricular Mass Independent of 24-Hour Brachial Pressures in a Community Sample
Authors: Woodiwiss Angela J , Sibiya Moekanyi J , Libhaber Elena , Libhaber Carlos , Booysen Hendrik L , Millen Aletta , Maseko Muzi J , Brooksbank Richard , Dessein Patrick H , Sareli Pinhas , Norton Gavin R
Published: March 2022

Whether 24-hour aortic as opposed to brachial blood pressure (BPb) adds to the ability of 24-hour BP to predict cardiovascular damage is uncertain. We imputed 24-hour central aortic pulse pressure (PPc) from an equation derived in 1179 participants. We showed that the relationship between the in-office PPc imputed using this equation and the radial pulse wave-derived PPc (SphygmoCor software) had an r2=0.96 and a mean difference (±2xSD) of -1.4±6.2 mm Hg. In 485 participants 24-hour brachial systolic BP (SBP), imputed PPc and the aortic-to-brachial amplification ratio (PPb/PPc) were associated with echocardiographic left ventricular mass indexed to height2.7 (LVMI) (r=0.28, r=0.37 and r=-0.39 respectively, p<0.0001 for all). However, the unadjusted relationship between 24-hour PPb/PPc and LVMI was stronger than that between 24-hour SBP and LVMI (p<0.05 for comparison of relations). In addition, the effect size (standardized β-coefficient±SEM) of the unadjusted relations with LVMI were greater (p<0.05) for 24-hour PPb/PPc (β=-0.40±0.04), than for 24-hour SBP (β=0.28±0.04). Twenty-four hour PPb/PPc and 24-hour PPc were associated with LVMI independent of 24-hour SBP (r=-0.30, p<0.0001; r=0.24, p<0.0001 respectively), and the strength and effect size of the unadjusted relations with LVMI were greater (p<0.005) for 24-hour PPb/PPc (β=-0.34±0.05) than for 24-hour SBP (r=0.11, p<0.05; β=0.12±0.05). Marked differences in LVMI (mean±SD in g/m2.7) were noted across quintiles of 24-hour PPb/PPc even with adjustments for confounders including 24-hour SBP (quintiles 1: 48.2±17.3; 2: 42.7±14.1; 3: 42.4±13.2; 4: 41.4±13.7; 5: 39.6±16.9; p<0.01 for quintile 1 versus quintiles 2-5). With adjustments including 24-hour SBP, quintile 1 of 24-hour PPb/PPc, showed an 8.6 g/m2.7 higher LVMI than quintile 5 (p<0.005). Importantly, the strength and effect size of the unadjusted correlations with LVMI were lower (p<0.05) for the in-office PPb/PPc derived from radial pulse wave analysis (r=-0.25, p<0.0001; β=-0.24±0.04, p<0.0001) than for 24-hour PPb/PPc (r=-0.39, p<0.0001; β=-0.40±0.04, p<0.0001). In conclusion, 24-hour PPb/PPc derived from imputed aortic BP values identifies LVMI better that does 24-hour BP or the in-office PPb/PPc.
Abstract 44: Angiotensin-(1-9) Decreases Hypertension and Vascular Damage by Inhibiting Rhoa/rock Activity in Doca Hypertensive Rats
Authors: Ocaranza Maria Paz , Villegas Bélgica , Moya Jackeline , Barrientos Victor , Novoa Ulises , Pérez Olga , Walkowiak Stefan , Chiong Mario , Lavandero Sergio , Michea Luis , Jalil Jorge
Published: March 2022

Previously, we found in experimental hypertension (DOCA salt model), that the RhoA/Rho-kinase pathway inhibition by fasudil decreases hypertension (HT), angiotensin (Ang) II levels and vascular remodeling. Besides, in this model fasudil increased ACE2 activity and Ang1-9 plasma levels. These results strongly suggest that in this experimental model, ROCK activity is more dependent on ACE2 and Ang-(1-9) than Ang II levels. There is no current evidence regarding the effect of Ang1-9 on ROCK activity of hypertensive rats.

Aim:
To determine the effect of the chronic administration of Ang1-9 on blood pressure (BP), vascular damage and activation of RhoA/ROCK pathway in deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

Methods:
The DOCA-salt hypertensive model was used in Sprague-Dawley male rats (age: 5-6 weeks; 150±10 g). DOCA was administered twice a week (60 mg/kg, im) starting immediately after surgery. The animals also received 1% NaCl and 0.4% KCl in the drinking water. Controls (C) were uninephrectomized rats (Sham group, n=12). Hypertensive rats (D) were randomized to receive vehicle (n=10), Ang-(1-9) (600 ng/kg/min, n=12), Ang-(1-9) + the Mas blocker receptor A779 (100 ng kg−1 min−1,n=7) and Ang-(1-9) + the AT2R antagonist
PD123319
(PD, 36 ng/Kg min) for 2 weeks. Systolic (S) and diastolic (D) BP, body mass (BM), heart weight (HW), heart mass relative to the length of the tibia (HMR), lumen area (LA), vascular reactivity assays (VRA % dilation at 10-9M AcCh) and ROCK activity by the ratio of phosphorylated vs total MYPT1 (MP/ MT) in the aortic wall were determined.

Results:
Compared to sham rats, in hypertensive rats VRA was significantly decreased (C: 15 ± 1.8 vs D: 2 ± 0.8), whereas SBP (C: 125 ± 2 vs D: 207 ± 7), AMT/LA (C: 0.32 ± 0.01 vs D: 0.4 ± 0.01) and MP/ MT (C: 1 ± 0.12 vs D: 1.77 ± 0.38) were significantly increased. In DOCA hypertensive rats, Ang1-9 significantly reduced SBP (161 ± 6), AMT/LA (0.37 ± 0.01) and aortic MP/ MT (0.65 ± 0.07) but increased VRA (25 ± 1.1). These beneficial effects of Ang-(1-9) were modified by
PD123319
but not by A779.

Conclusion:
Ang1-9 reduces BP and vascular damage by inhibiting ROCK activity in this model of low renin HT. These effects seem be mediated by the AT2R. Fondecyt 1100874 and 1121060.
Abstract 443: Combining Irbesartan and Trichlormethiazide Elicits Strong Blood Pressure Reduction Associated with Sympathoinhibition and Beneficial Metabolic Profiles in Hypertensive Rats with Metabolic Syndrome
Authors: Nishihara Masaaki , Hirooka Yoshitaka , Sunagawa Kenji
Published: March 2022

Background:
Sympathoexcitation accompanied by obesity plays a pivotal role in hypertension associated with metabolic syndrome which is characterized by the presence of central obesity, impaired fasting glucose, and dyslipidemia. We have demonstrated that the oxidative stress in the rostral ventrolateral medulla (RVLM) increases sympathetic activity in hypertensive rats. The aim of the present study was to determine whether orally administered angiotensin II receptor blocker (irbesartan; IRB) and diuretics (trichlormethiazide; TCM) decreases blood pressure (BP) via inhibiting sympathetic activity through anti-oxidant effects in the RVLM of spontaneously hypertensive/NDmcr-cp rats (SHR-cp) without eliciting sympathoexcitation due to the strong antihypertensive action. We also investigated the effects of IRB/TCM therapy on the metabolic profile.

Methods and Results:
IRB (25 mg/kg/day), TCM (1 mg/kg/day), IRB/TCM (25/1 mg/kg/day) or vehicle; VEH were orally administered for 28 days to SHR-cp (from age of 12 week-old). Each treatment decreased systolic BP (SBP) compared with VEH group (SBP after treatment; IRB 132±1, TCM 147±2, IRB/TCM 118±2 vs. 162±4 mmHg,
P
<0.01, n=6~7), and IRB/TCM treatment decreased SBP to a greater extent than IRB monotherapy. Both urinary norepinephrine excretion (uNE) as a marker of sympathetic activity and oxidative stress evaluated by thiobarbituric acid-reactive substances (TBARS) levels in the RVLM were decreased in both IRB and IRB/TCM groups compared with VEH group (uNE; 1.1±0.1 and 1.4±0.1 vs. 2.0±0.1μg/day,
P
<0.01, n=6~7), (TBARS; 0.52±0.02 and 0.54±0.02 vs. 0.73±0.03μmol/g wet wt,
P
<0.01, n=6~7), but not in TCM group. IRB/TCM treatment did not exert an adverse effect on the metabolic profile, such as fasting blood glucose, total cholesterol, LDL-cholesterol, and HDL-cholesterol, but it decreased the triglyceride levels of SHR-cp compared with VEH group (553±40 vs. 730±37mg/dl,
P
<0.01, n=6 for each).

Conclusions:
These findings suggest that the IRB/TCM combination therapy have a strong antihypertensive effect associated with inhibiting the sympathetic activity reducing oxidative stress in the brain and

preferable metabolic profiles in SHR-cp.
Abstract 456: Assessment of Pharmacokinetic Drug Interaction between LCZ696 and Metformin
Authors: Mendonza Anisha , Akahori Mizuki , Langenickel Thomas , Swan Therese , Pal Parasar , Sunkara Gangadhar , Chandra Priyamvada
Published: March 2022

Objective:
LCZ696 is a first-in-class angiotensin receptor neprilysin inhibitor (ARNI) being developed for the treatment of cardiovascular diseases, including hypertension and heart failure. Ingestion of LCZ696 results in systemic exposure to AHU377 (inactive prodrug that is converted to LBQ657, a neprilysin inhibitor) and valsartan (angiotensin receptor blocker). Metformin is the most commonly prescribed oral treatment for type 2 diabetes mellitus (T2DM), a condition prevalent in patients with cardiovascular diseases. Since LCZ696 and metformin may be co-administered, the potential for a pharmacokinetic (PK) interaction was assessed between these drugs.

Methods:
The study was conducted in 27 healthy subjects of Japanese descent using an open-label, three-period, single-sequence design. In Period 1, subjects received metformin 1000 mg once daily (q.d.) for 4 days. Following a metformin washout of 4-10 days, subjects received LCZ696 400 mg q.d. for 5 days in Period 2. In Period 3, LCZ696 400 mg q.d. and metformin 1000 mg q.d. were co-administered for 4 days. Serial PK samples were taken during all treatment periods and analyzed using validated LC/MS/MS bioanalytical techniques. The PK parameters (Cmax,ss, AUCtau,ss) of active LCZ696 analytes (LBQ657 and valsartan) and metformin were determined using non-compartmental analysis, and the results were evaluated by statistical analysis.

Results:
The 90% confidence intervals for the geometric mean ratios for Cmax,ss and AUCtau,ss of LCZ696 analytes (LBQ657 and valsartan) were within the 0.8-1.25 range indicating that metformin did not affect the PK of the LCZ696 analytes. The lower bound of the 90% confidence intervals for the geometric mean ratios for metformin exposures were below the 0.8-1.25 range (Cmax,ss: 0.77, 0.70-0.84; AUCtau,ss: 0.77, 0.71-0.82), indicating a decrease in Cmax,ss and AUCtau,ss of metformin by 23%.

Conclusion:
After co-administration of LCZ696 400 mg q.d. and metformin 1000 mg q.d., the exposures of LBQ657 and valsartan remained unchanged whereas the exposure to metformin decreased by 23%. LCZ696 400 mg q.d. was safe and well tolerated in healthy subjects when administered alone and in combination with metformin 1000 mg q.d..
Abstract 464: Molecular and Functional Interaction of the Angiotensin AT2-Receptor and the Receptor MAS
Authors: Villela Daniel , Leonhardt Julia , Muenter Lisa M , Lucht Kristin , Benz Verena , Namsolleck Pawel , Alenina Natalia , Unger Thomas , Bader Michael , Santos Robson A , Steckelings Ulrike M
Published: March 2022

The angiotensin AT2-receptor (AT2R) and the receptor MAS both mediate very similar tissue-protective actions. Furthermore, AT2R-antagonists have been reported to block effects of the MAS-agonist Ang 1-7. Therefore we speculated that a molecular association between these two receptors exists resulting in functional interactions.

Fluorescence resonance energy transfer (FRET) with photobleaching was used for detection of a putative interaction between MAS and AT2R in HEK-293 cells transfected with vectors encoding MAS or AT2R fused in the C-terminus with CFP or YFP. In addition, functional interaction of AT2R and MAS was assessed in primary mouse astrocytes by determining the effect of respective agonists and antagonists on expression of mRNA encoding the chemokine receptor CX3CR1.

There was significant FRET efficiency of 10.8±0.8% when AT2-YFP and MAS-CFP were co-expressed indicating heterodimerisation. Both, MAS and AT2R also formed homodimers (FRET efficiencies: 7.4±0.8% or 9.2±0.8%, respectively). No FRET efficiency was observed with an unrelated transmembrane receptor, and expression of non-fluorescent MAS and AT2R competed with FRET efficiencies, which both supports specificity of receptor interactions. Regarding putative functional interactions, AT2R-agonist induced increases in CX3CR1 mRNA expression were absent in astrocytes concomitantly treated with the MAS-antagonists A-779 or in Mas-deficient cells. Likewise, effects of the Mas agonist Ang 1-7 were undetectable in cells concomitantly treated with the AT2-antagonist
PD123319
or in AT2-deficient cells.

This study provides evidence that MAS and AT2 receptors form homo- and heterodimers. Heterodimerisation may be of functional relevance, because in primary mouse astrocytes, Mas and AT2 seemed only functional in the presence of the respective other receptor.
Abstract 471: Mechanism of Pressure-Natriuresis: Evidence that Extracellular cGMP Signals through Renal Basolateral Na+ K+-ATP-ase
Authors: Kemp Brandon A , Gherghe Cristina , Howell Nancy L , Carey Robert M
Published: March 2022

Pressure-Natriuresis (P-N) is a major compensatory mechanism that prevents an increase in blood pressure by increasing renal sodium excretion (U
Na
V). Our group showed that cGMP, at least in part, mediates P-N. cGMP signals from the extracellular space through a yet unidentified receptor in the renal proximal tubule basolateral membrane. We hypothesize that cGMP binds to Na
+
K
+
ATP-ase (NKA), activating both the Src kinase and the EGFR-Erk-Akt signaling cascades.

In uninephrectomized rats, the renal perfusion pressure (RPP) was increased from 100 to 135 mmHg. During high RPP, U
Na
V increased 6.3-fold and the ratio of phosphorylated Src (Tyr
416
)/total Src (pSrc) increased from 0.07±0.02 to 0.71±0.12 μmol/min (P<0.001, N=5 for both). Blocking the NKA ouabain (OUA) binding site by infusing rostafuroxin (RF, a specific OUA antagonist without effect on NKA pump activity) directly into the renal interstitium reduced U
Na
V 6.4-fold and pSrc to 0.22±0.05 (N=8, P<0.05). Both phosphorylation of Erk 1/2 (p44/42 MAPK) and Akt (Ser
473
) were increased during the high RPP period (0.35±0.07 to 2.08±0.21; N=6 for both, P<10
-4
and 0.39±0.06 to 1.04±0.15; N=8 for both, P<0.01, respectively). However, RF did not decrease pErk and pAkt, likely due to
in vivo
compensatory processes following NKA blockade.

To delineate the role of cGMP in P-N, we infused cGMP into the renal interstitium. U
Na
V increased 5.7-fold and pSrc increased from 0.09±0.02 (N=4) to 0.35±0.1 (N=6, P<0.05). OUA infusion was used as a positive control and increased pSrc to 0.77±0.01 (N=4, P<10
-6
). Coinfusion of cGMP and RF reduced pSrc to 0.15±0.03 (P=0.07, N=6). Surprisingly, RF infusion also decreased pErk from 0.71±0.17 (N=6, in cGMP-infused rats) to 0.33±0.09 (N=6, in cGMP and RF-coinfused rats, P=0.085).

In summary, renal interstitial infusion of an NKA signaling antagonist RF blocks Src activation during both P-N and cGMP-induced natriuresis. P-N-induced Erk and Akt activation is not affected by RF. However, Erk and Akt phosphorylation is reduced during cGMP-induced natriuresis, suggesting a possible competition between cGMP and RF for the same binding site on NKA. The data support the hypothesis that P-N is mediated by extracellular cGMP binding to basolateral NKA, which serves as a receptor.
Abstract 48: Targeting the Endothelial Progenitor Cell Surface Proteome to Identify Novel Mechanisms that Mediate Angiogenic Efficacy and Restore Angiogenesis in a Rodent Model of Vascular Disease
Authors: Kaczorowski Catherine , Stodola Tim , Hoffmann Brian , Prisco Anthony , Lui Pengyuan , Didier Daniela , Karcher Jamie , Liang Mingyu , Jacob Howard , Greene Andrew
Published: March 2022

Bone-marrow derived endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials suggest autologous EPC-based therapy may be effective in treatment of vascular diseases. Albeit promising, variability in the efficacy of EPCs associated with underlying disease states has hindered the realization of EPC-based therapy. Here we identify and characterize EPC dysfunction in a rodent model of vascular disease (SS/Mcwi rat) that exhibits impaired angiogenesis under physiological conditions, and develops cardiovascular disease on a high salt diet. First, we compared the angiogenic responses to EPCs delivered into the skeletal muscle of SS/Mcwi recipient prior to electrical stimulation. Delivery of EPCs from SS/Mcwi donors had no effect on angiogenesis compared to stimulation alone (9.2± 2.2% vs. 7.6 ± 1.7%, respectively). In contrast, introgression of chromosome 13 from a Brown Norway rat onto the SS/Mcwi background (SS-13
BN
/Mcwi rat) significantly enhanced the angiogenic function of EPCs (22.3 ± 3.7%) and suggests involvement of genes on chromosome 13. To identify molecular candidates that mediate the angiogenic potential of these cells, we performed a cell surface proteomic analysis. Analysis revealed that EPCs derived from SS/Mcwi rats express significantly more type 2 low-affinity immunoglobulin Fc-gamma (FCGR2, 25% increase) and Natural Killer 2B4 (CD244, 67% increase) receptors than EPCs from the SS-13
BN
/Mcwi rat. Genome-wide mRNA sequencing (RNAseq) revealed differential expression of multiple isoforms encoding FCGR2a and CD244 proteins, and qt-PCR confirmed an increase in CD244 and FCGR2a transcripts in SS/Mcwi EPCs. Increased expression of FCGR2a and CD244 receptors are predicted to increase the probability of SS/Mcwi EPCs being targeted for death, providing a mechanistic explanation for their reduced angiogenic efficacy
in vivo
. Pathway analysis supported this contention, as ‘key’ molecules annotated to cell death paths were differentially expressed in the SS/Mcwi EPC transcriptome. We speculate that screening and neutralization of cell surface proteins that tag ‘diseased’ EPCs for death will enhance regenerative potential of EPC-based therapies, providing a major advance in the field of regenerative medicine.
Abstract 481: Age-related Hypertension and Salt-sensitivity Are Associated With Specific Kidney Cortico-medullary Distribtion of D1R, AT1R And NADPH-oxidase
Authors: Pokkunuri Indira
Published: March 2022

We recently reported that oxidative stress has a causal role in increasing kidney angiotensin II AT1 receptor (AT1R) and decreasing dopamine D1 receptor (D1R) functions in the aging Fischer Brown Norway (FBN) rats. These renal receptors by counter-regulating each other’s functions maintain sodium homeostasis and normal blood pressure (BP). Here, we examined systolic BP and kidney distribution (cortex vs medulla) of these receptors as well as of an oxidant producing enzyme (gp
91phox
-NADPH-oxidase) in response to 4-week normal-salt (NS, 0.4% NaCl) and high-salt (HS, 8% NaCl) feeding in adult (3-month) and old (21-month) FBNs. BP in conscious animals was measured by radio-telemetry. Distribution of D1R, AT1R and gp
91phox
were determined by RT-PCR and western blotting. We found that NS-fed old rats had higher BP (adult vs old: 105±1 vs 118 ±1 mmHg), which further increased with HS feeding in these rats (adult vs old: 111±1 vs 124 ±1 mmHg). The levels of D1R mRNA in cortex or in medulla were not different between NS-fed adult and old rats, but decreased in both cortex [adult vs old: 67 ± 15 vs 54±16 density unit (DU)] and medulla (adult vs old: 22±15 vs 11±3 DU) only in HS-fed old rats. Contrary to this, AT1R mRNA levels were higher only in cortex of old rats fed either NS (adult vs old: 81 ± 23 vs 102± 34 DU) or HS (adult vs old: 80 ± 17vs 98 ± 36 DU). Moreover, there were similar gp
91phox
mRNA levels in the cortex and medulla of adult and old rats fed either NS or HS. But, its protein levels were higher in the cortex of old rats fed NS (adult vs old: 60±7 vs 112±8 DU) and HS (adult vs old: 61±3 vs 94±15 DU), and increased only in the medulla with HS feeding in these rats (adult vs old: 12±1 vs 28±2 DU). Our results suggest that there exists a specific cortico-medullary expression pattern of D1R, AT1R and gp
91phox
with NS and HS feeding in old rats, which might be contributing to age-related hypertension and salt-sensitivity in FBNs.