bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 610: Context-Dependent Effects of Suppressor of Cytokine Signaling 3 (SOCS3) in Angiotensin II-Induced Vascular Dysfunction: Mechanisms and Role of Bone Marrow-Derived Cells
Authors: Li Ying , Kinzenbaw Dale , Modrick Mary , Epping Lecia , Harty John T , Faraci Frank M
Published: July 2021

Angiotensin II (Ang II) promotes vascular disease and hypertension, in part, by activating the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway. Although SOCS3 regulates this pathway in the immune system, its role in vascular disease and hypertension is unknown. In this study, we investigated the role of SOCS3 in a model of Ang II-induced vascular disease. To exam direct effects, carotid arteries from wild-type (WT) and SOCS3 haplodeficient (SOCS3
+/-
) mice were incubated with Ang II for 22 hrs, followed by examination of endothelial function using acetylcholine (Ach). Relaxation to Ach was similar in all arteries incubated with vehicle. A low concentration of Ang II (1 nmol/L) did not affect Ach-induced vasodilation in WT mice, but reduced that of SOCS3
+/-
mice by ~50% (P<0.05). Ang II-induced impairment was prevented by inhibitors of STAT3, IL-6, NF-κB, or a scavenger of superoxide. Responses to nitroprusside were similar in all groups. We also tested the impact of SOCS3 in vivo by systemically infusing Ang II (1.4 mg/kg per day) for 14 days via osmotic mini-pumps. Ach-induced vasodilation in carotid and resistance arteries in brain from WT mice was reduced by ~60% (P<0.05). Surprisingly, deficiency in SOCS3

prevented the majority of Ang II-induced endothelial dysfunction without affecting the pressor response to Ang II. Lethally irradiated WT mice reconstituted with SOCS3
+/-
bone marrow were protected from Ang II-induced endothelial dysfunction (P<0.05), while reconstitution of irradiated SOCS3
+/-
mice with WT bone marrow exacerbated Ang II-induced vascular dysfunction (P<0.05). WT into WT and SOCS3
+/-
into SOCS3
+/-
bone marrow chimeras exhibited vascular function consistent with non-irradiated controls. The pressor response to Ang II was reduced by ~50% in WT mice reconstituted with bone marrow from SOCS3
+/-
mice (P<0.05). These data suggest SOCS3 exerts divergent local versus systemic effects on Ang II-induced vascular dysfunction. In the face of SOCS3 deficiency, bone marrow-derived cells protect against Ang II-induced vascular dysfunction and hypertension.
Abstract 612: ACE2-Independent Sex Differences In Oxidative Stress
Authors: Wysocki Jan , Evora Karla , Salem Moody , Maier Christoph , Ye Minghao , Batlle Daniel
Published: July 2021

Many of the pathophysiological effects of angiotensin II (Ang II) are attributed to its stimulation of NADPH oxidases and the consequent production of reactive oxygen species. Female sex has generally lower cardiovascular morbidity and is also less susceptible to kidney injury than males. The basis of these phenomena is not well understood but it is possible that sex-differential regulation of oxidative stress through activation of the RAS and its key effector, Ang II, plays an important contributor role. Here we hypothesized that Ang II levels are higher in male mice and that this is associated with sex-differences in kidney levels of ACE2, an Ang II-degrading enzyme abundantly expressed in the kidney.

Parameters of oxidative stress such as, NADPH oxidase activity and malondialdehyde levels (MDA) were measured in kidneys from female and age-matched male C57BL6 mice. At 40 weeks of age, NADPH oxidase activity (p<0.01) and MDA levels (p<0.05) were significantly lower in female than in male mice. Female mice had lower kidney levels of the pro-oxidant peptide, Ang II (0.94±0.19 vs. 1.66±0.17 fmol/mg total protein, p<0.05, respectively). The difference in kidney Ang II levels between females and males was also observed in the face of complete
ACE2
genetic deficiency (1.08±0.16 vs 1.97±0.25 fmol/mg total protein, p<0.05, respectively). Consistent with kidney Ang II levels, urinary Ang II levels measured in urines from female WT mice were also significantly lower than in male WT mice (23.6±2.2 vs. 47.9±8.8 pg/mg creatinine, p<0.05) despite significantly higher levels of urinary ACE2 activity in male mice as compared to female mice (7.0±0.5 vs. 3.6±0.3, p<0.01, respectively).

Female mice have lower basal levels of kidney oxidative stress than males and exhibit lower levels of kidney and urinary Ang II. The mechanism involved in sex differences in the levels of kidney and urine Ang II does not appear to depend on ACE2.
Abstract 626: Interaction Between The Ang-(1-7) Receptor Mas And The Bradykinin B2 Receptor: Functional Implications
Authors: Cerrato Bruno , Carretero Oscar , Grecco Hernán , Gironacci Mariela M
Published: July 2021

G protein-coupled receptors (R) exist as homo- or hetero-oligomers, which is essential for receptor function. Since BK actions were blocked by a Mas R antagonist or that Ang-(1-7) responses disappeared when the BK receptor B2 was blocked, we hypothesized that Mas and B2 Rs on the plasma membrane may interact through hetero-oligomer formation. Our aim was to investigate the existence of heteromerization between Mas and B2 Rs by the fluorescence energy transfer (FRET) technique and the functional consequences of this oligomer formation. HEK293T cells were transfected with the coding sequence for Mas R fused to YFP and B2 R fused to CFP. After 48 h cells were incubated in the absence and presence of 1 μM Ang-(1-7) or BK during 15 min and interaction between Mas and B2 R was evaluated by FRET. Functional consequences of this interaction were determined by ligand binding assays. A positive FRET was observed in cells cotransfected with MasR-YFP and B2R-CFP, suggesting that both Mas and B2 Rs interact by a hetero-oligomer formation in a constitutive manner. This hetero-oligomer was not altered by the agonist because FRET was not modified when the cells were stimulated with BK or Ang-(1-7). Ang-(1-7) or BK induced internalization of this hetero-oligomer into early endosomes since MasR-YFP or B2R-CFP colocalized with Rab-5, an early endosome marker, after ligand stimulation. When MasR-YFP plus B2R-CFP transfected cells were stimulated with Ang-(1-7) there was a decrease of 82±6% in Mas R and 58±4% in B2 R present in the plasma membrane. Conversely, when MasR-YFP plus B2R-CFP transfected cells were stimulated with BK there was a decrease of 91±4% in B2 R and 53±3% in Mas R in the plasma membrane. This result clearly demonstrates that in co-expressing cells of both receptors the selective stimulation of one of the GPCRs promotes co-internalization of both receptors. We conclude that Mas and B2 Rs constitutively interact through an hetero-oligomer formation at the plasma membrane which may explain the cross-talk between Ang-(1-7) and BK. This hetero-oligomer is internalized upon stimulation with either Ang-(1-7) or BK, leading to a decrease in the number of Rs present in the membrane.
Abstract 632: Vasomera, a Novel VPAC2-Selective Vasoactive Intestinal Peptide Agonist, Improves Arterial Elastance and Ventriculo-Arterial Coupling in Rats with Induced Diastolic Dysfunction via Renoprival Hypertension
Authors: del Rio Carlos L , Ueyama Yukie , Georgopoulos Lynne , Arnold Sue , Hamlin Robert L
Published: July 2021

Vasomera™ is a first-in-class stable long-acting vasoactive intestinal peptide (VIP) agonist, with preferential actions on the G-protein-coupled VPAC2-receptors; VIP mediates cardiopulmonary regulation and has been proposed as a therapeutic target for both hypertension and systolic dysfunction. Here, the acute effects of Vasomera in load-independent function and ventriculo-arterial coupling were in evaluated in a rats with chronic diastolic dysfunction, mimicking heart failure with preserved ejection fraction (HFpEF).

Diastolic dysfunction, as demonstrated by altered E/A ratios via echocardiography, was induced by renoprival hypertension secondary to renal wrapping (RW). Conditioned rats (n = 5) were instrumented for the determination of left-ventricular (LV) hemodynamics as well as load-independent fnction and ventriculo-arterial coupling (via pressure-volume relationships); data were evaluated before/after a single-dose continuous IV infusion of Vasomera (7.5 μg/kg/min IV).

Vasomera decreased the estimated arterial elastance (Ea: -20 ± 5%, P < 0.05) with negligible changes in heart rate (-4 ± 2%). Improved inotropy (Ees: +28 ± 8% and PRSW: +22 ± 3%, P < 0.05) was observed post-treatment, suggesting improved ventriculo-arterial coupling (Ea/Ees: -37 ± 5%, P < 0.05). Concomitantly, Vasomera reduced filling pressures (EDP: -30 ± 8%, P < 0.05), accelerated the time-constant of relaxation (tau: -24 ± 3%, P < 0.05) and improved compliance (EDPVR: -26 ± 5%, P < 0.05).

Vasomera, a novel VPAC2 agonist, improved arterial elastance and ventriculo-arterial coupling, while improving indices of diastolic function (i.e., lusitropy) in animals with chronic renoprival hypertension mimicking HFpEF.
Abstract 650: Mechanism Underlying The Increased Contractile Sensitivity In Serum-free Organ-cultured Rat Mesenteric Artery
Authors: Morita Tomoka , Okada Muneyoshi , Yamawaki Hideyuki
Published: July 2021

Background:
Organ culture of blood vessels in a serum-free condition is a better way to maintain differentiated cell function. However some functional changes may occur from freshly isolated blood vessel (Fresh). The removal of proteins (enzymes and regulatory proteins) is essential for the control of cell growth and metabolism. Failure to remove an essential protein or lack of degradation of a short-lived regulatory protein may alter cell function in a major way. We thus hypothesized that the ubiquitin proteasome system may be a candidate which causes functional changes. The aim of the present study was to investigate the effects of an inhibitor of ubiquitin proteasome on increased contractile sensitivity in serum-free organ-cultured rat mesenteric arteries.

Methods and results:
Rat isolated mesenteric arteries with endothelium were cultured for 5 days without (0% serum) or with an ubiquitin proteasome inhibitor, Bortezomib (Bort; 10 nM). In 0% serum, sensitivity to KCl (5.4-75.4 mM) and phenylephrine (0.01-100 uM) significantly increased from Fresh (KCl: EC50 (mM) for 0% serum and Fresh was 18.3 and 40.3, respectively, p<0.01) (phenylephrine: pD2 for 0% serum and Fresh was 6.58 and 5.07, respectively, p<0.01), while maximal contraction significantly decreased. In Bort, the increased sensitivity to KCl and phenylephrine was significantly normalized (EC50 for Bort was 24.9 (mM) and pD2 for Bort was 3.99, respectively, p<0.01), while maximal contraction did not change from 0% serum. A rho-associated kinase inhibitor, Y27632-induced relaxation in Bort significantly decreased from 0% serum, while relaxation induced by aβ-adrenergic receptor agonist, isoproterenol, a phosphodiesterase 3 inhibitor, cilostazol, a protein kinase C inhibitor, Go6983 and a phospholipase C inhibitor, U73122 did not change between 0% serum and Bort.

Conclusion:
These data suggest that ubiquitin proteasome system increases contractile sensitivity via a rho-associated kinase in serum-free organ cultured mesenteric artery. Elucidation of the mechanism might contribute to provide mechanistic insights into the role of ubiquitin proteasome system on pathogenesis of hypertension.
Abstract 652: dTGR: A New Rat Model For Systemic Hypertensive Retinopathy
Authors: Reichhart Nadine , Haase Nadine , Herrspiegel Christina , Müller Dominik N , Crespo-Garcia Sergio , Skosyrski Sergej , Dechend Ralf , Strauß Olaf
Published: July 2021

Retinal vascular alterations represent an important but understudied form of microvascular target endorgan damage (TOD). To understand the pathomechanisms underlying hypertensive retinopathy, we used a double transgenic rat model (dTGR) established for TOD, which expresses both the rat and human RAS. Purpose of the study is to assess whether systemic hypertension affects retinal structure and function similar to the characteristic pathology in humans.

We investigated the retinal phenotype by using immunohistochemistry of retinal whole mount preparations or sagittal sections, qPCR as well as functional analysis by Ganzfeld and pattern electro-retinography (ERG).

Whole mount preparations revealed focal avascular areas, pathological vessels with irregularities in shape and diameter as well as neovascular tufts in dTGR rats compared to control animals. In sagittal sections we detected a loss of retinal ganglion cells. GFAP staining revealed activation of astrocytes in the ganglion cell layer (GCL) and astrocytosis of the optic nerve. VEGF-positive staining could be detected in the ganglion cell layer (GCL) and the inner nuclear layer (INL).

To further substantiate the loss of ganglion cells detected in immunohistochemistry and to obtain a correlated functional analysis, pattern ERG were recorded. By using a stimulation with different pattern sizes (15° and 5-7°) at 2.4hz of the black and white checkerboard we showed that the positive component after about 50ms (P50) of the pattern ERG was significantly smaller in dTGR compared to control rats at 5-7° pattern size whereas the P50 at 15° remained unchanged. This indicates a loss of ganglion cells in the retina.

qPCR revealed a non-detectable local renin expression, increase in MCP1 and sFLT1 expression and decrease in the expression of tight-junction proteins claudin-1 and occludin.

Thus we present a first animal model for hypertensive retinopathy showing functional and structural alterations of the retina. Differential gene expression analysis suggests a strong local inflammatory response, loss of the blood/retina barrier and surprisingly low activity of the local renin-angiotensin-system. Further studies are warranted to investigate the local pathomechanisms.
Abstract 655: Dipeptidyl Peptidase-4 (dpp-4) Inhibition Decreases Cardiac And Vascular Stiffness And Improves Cardiac And Vascular Relaxation In Western Diet Fed Mice
Authors: Aroor Annayya R , DeMarco Vincent G , Jia Guanghong , Martinez-Lemus Luis A , Habibi Javad , Sun Zhe , Garro Mona , Meininger Gerald A , Sowers James R
Published: July 2021

A western diet (WD), high in sucrose and fat, is often accompanied by insulin resistance and cardiovascular disease characterized early by endothelial dysfunction and increased vascular and cardiac stiffness. Recently, Dipeptidyl peptidase-4 (DPP-4) inhibition has been shown to improve diastolic dysfunction in WD fed mice, but its effects on endothelial cell and cardiac stiffness have not been reported. We fed 4-week old C57BL/6 male mice with a WD with or without a DPP-4 inhibitor (MK0626) for 16 weeks and measured blood pressure by telemetry, insulin resistance via (HOMA), in vivo cardiac diastolic function (echocardiography), pulse wave velocity (PWV), and ex vivo aortic endothelial stiffness by atomic force microscopy. Systolic blood pressure and insulin resistance were increased by the WD. DPP-4 inhibition improved systemic insulin sensitivity and substantially reduced DPP-4 activity, but had no effect on 24-hour blood pressures. Heart weight was increased by WD in conjunction with S6 kinase translational signaling and DPP-4 inhibition reduced S6 kinase phosphorylation/activation in conjunction with a reduction in cardiac mass. Aortic stiffness, as assessed by PWV, was significantly increased in WD fed mice (16% increase) and was markedly decreased by DPP-4 inhibition. Endothelial cell stiffness was increased 5-fold in WD fed mice and DPP-4 inhibition significantly decreased endothelial stiffness (80% decrease). Acetylcholine but not sodium nitroprusside mediated vascular relaxation was impaired in WD fed mice and DPP-4 inhibition significantly improved this nitric oxide mediated relaxation. Increased vascular smooth muscle and endothelial stiffness was associated with impaired cardiac diastolic relaxation, which was also significantly improved by DPP-4 inhibition. Taken together, these results show that DPP-4 inhibition improves cardiac and vascular endothelial stiffness and cardiac diastolic dysfunction in a clinically translational mouse model (WD) of over nutrition and insulin resistance.
Abstract 658: New Onset Diabetes Does Not Pose Additional Risk - Analysis Of 15,111 Treated Hypertensive Patients With 40 Year Follow-up.
Authors: Lip Stefanie
Published: July 2021

Objective:
It is unclear whether new onset diabetes (NOD) is a separate entity associated with excess risk in hypertensive patients.

Methods:
We studied 15111 hypertensive patients with up to 40 year follow-up at the Glasgow BP Clinic database. Diabetes status was defined based on hospital admissions for any diabetes related diagnosis or prescription of anti-diabetic drugs or diabetes monitoring materials. The date at first hospital encounter either for prescription or admission was considered as the onset of diabetes. NOD was classified into early and late (diagnosis <10yrs or >10years from first clinic visit). Cause-specific outcome analysis was performed using multivariate-adjusted Cox proportional hazards (Cox-PH) models. In order to address any potential competing risk introduced due to the long follow-up period, an additional composite end point of all-cause mortality+NOD was analysed.

Results:
There were 2521(17%) patients with DM, of whom 2061(14%) had NOD. The incidence rate of NOD was 9.2 per 1000 person-years. Prevalence of early NOD was 898 (6%) and late NOD 1163 (8%). The total time at risk was 239,952 person-years with a median survival time of 28.04 years (IQR: 16.24-39.95). There were 5225 deaths (52% from cardiovascular causes) during the follow-up period. Independent predictors of new-onset diabetes in order of decreasing significance were baseline glucose, BMI, age, alanine transaminase, alkaline phosphatase, gamma glutamyl transpeptidase and bilirubin. Of these age, glucose, BMI and alkaline phosphatase remained top predictors for the composite outcome of NOD+all-cause death. The mortality risk was the highest in those with prevalent DM (HR=1.5[95%CI=1.2;1.9]) and lowest in those with late NOD (0.79[0.68;0.92]). Early NOD and non-diabetic subjects had similar risks.

Conclusions:
Indices of liver function tests predict the risk of NOD and mortality in addition to BMI and baseline glucose. The risk posed by NOD is related to duration of diabetes primarily indicating the importance of efforts to delay onset of NOD.
Abstract 663: Metabolomics And Correlation Network Analysis Of Hypertension
Authors: tian zhongmin , wang le , hou entai , sun qiong
Published: July 2021

The awareness, treatment and controls rates of hypertension for people in their 20s and 30s age are much lower than average. In this paper, a GC/MS based metabolomics study was performed in plasma of young hypertensive men and age-matched normal ones. Correlations of the identified metabolites were analyzed and visualized. A systematic correlation network was constructed with the significance of correlation coefficient setting at threshold of 0.6. Glycine, lysine, cystine and beta-alanine were selected as the most important nodes of the network, with high values of degree. A relatively short average path length and high clustering coefficient suggested a small-world property of the network. Moreover, differential metabolites in young hypertensive men were used to construct a core correlation network for further understanding. Four hubs (lysine, glycine, cystine and tryptophan) were confirmed by a comprehensive evaluation of three centrality indices. The statistical and topological parameters of the network indicated that local disturbance to hubs would rapidly transfer to the whole network. These results demonstrated that the distinct metabolic profiles of young hypertensive men might be due to perturbation of the biosynthesis pathway of amino acids. Integrated analyses of metabolomics and correlation networks would provide a broadened window for further understanding of hypertension.

Key Words:
metabolomics; hypertension; correlation network; amino acids; statistical and topological characteristics; centrality indices; hubs
Abstract 665: Cryofluorescence 3D Imaging Shows Mutation of p67phox Improves Metabolic Function and Reduces Oxidative Stress in the Renal Medulla of the Dahl Salt-sensitive Rat
Authors: Cowley Allen W , Salehpour Fahimeh , Yang Chun , Kurth Theresa , Ranji Mahsa
Published: July 2021

The Dahl salt-sensitive (SS) rat exhibits increased renal production of reactive oxygen species (ROS) especially in the renal outer medulla (OM) which is known to contribute importantly to the salt-induced hypertension. We have identified increased expression of the p67
phox
cytosolic subunit of NAD(P)H oxidase and enhanced enzyme activity in the OM of Dahl salt-sensitive (SS). We found that ZFN knock down of the NADPH-oxidase cytosolic subunit p67
phox
in SS rats (SS
p67phox-/-
) resulted in a 40% reduction of hypertension and a substantial reduction of renal injury. To determine whether these protective effects were associated with alterations of regional metabolism and oxidative stress, a custom designed cryoimager was used to acquire multi-channel fluorescent images of sequential serial sections with a z-resolution of 30 μm yielding 400 slices per kidney. Computer reconstruction of the stacked sections provided a 3D image of regional changes of metabolic function and oxidative stress within the kidney. Kidneys obtained from SS and SS
p67phox-/-
rats fed a high salt diet (4% NaCl) for 21 days were flash frozen in liquid N
2
and fluorescent images of the mitochondrial electron transport chain carriers NADH and FAD were acquired. The naturally fluorescent NADH and FAD levels were acquired to provide a 3D representation of the metabolic state of the tissue and oxidative stress. The mean NADH redox ratio (NADH/FAD RR) was significantly higher in kidneys of SS
p67phox-/-
rats (1.46 ± 0.11 NADH/FAD RR; n=7) compared to SS kidneys (1.00 ± 0.07 NADH/FAD RR; n=4). This represents an average 46% increase in the electron transport chain metabolic activity and a reduction of oxidative stress in kidneys of SS
p67phox-/-
rats compared to the SS kidneys. Importantly, this was
observed only in the region of the renal medulla
as revealed by the 3D images of these kidneys. We conclude that p67(phox) is critically involved in cell energetics and ROS production in the renal medulla.
Abstract MP04: In Vivo Bioluminescence Imaging Reveals a Pre-Hypertensive Surge of Nuclear-Factor-κ-B (NF-κB) Activity in the Subfornical Organ (SFO) During Obesity-Induced Hypertension
Authors: Young Colin N , Butler Scott D , Davisson Robin L
Published: July 2021

The central nervous system has been implicated in obesity-induced sympathetic activation and hypertension, although the underlying mechanisms remain unclear. We tested the hypothesis that the transcription factor NF-κB is activated in the SFO, a key cardioregulatory brain center, during the development of obesity-induced hypertension. Male C57Bl/6 mice were instrumented with radiotelemeters for mean arterial pressure (MAP) recording, and underwent SFO-targeted microinjection of an adenovirus encoding firefly luciferase downstream of the NF-κB response elements. After recovery, mice were fed high fat diet (60% fat) or normal chow (5% fat), and SFO NF-κB activity was longitudinally monitored
in vivo
by bioluminescence imaging coupled with luciferin injections. In response to HFD, a slowly developing hypertension was observed, with an initial rise in MAP at 4 weeks (109 ± 1 vs 117 ± 1 mmHg; day 0 vs day 28; n=8; p<0.05) that was sustained for 8 weeks. NF-κB activity in the SFO increased gradually during HFD feeding, with a surge at 3 weeks, prior to a rise in MAP (day 21: 0.91 ± 0.08 vs 1.70 ± 0.25 photons/s fold baseline; normal chow vs HFD; n=4-7; p<0.05). SFO NF-κB activity transiently subsided and a second elevation was noted during the maintenance of obesity-induced hypertension at 6.5 weeks (day 42: 0.72 ± 0.14 vs 1.52 ± 0.07 photons/s fold baseline; normal chow vs HFD; n=4-7; p<0.05). These findings suggest that NF-κB is activated in the SFO during HFD feeding. The biphasic increase in NF-κB activity prior to and during a sustained increase in MAP suggests a casual role for SFO NF-κB in the development of obesity-induced hypertension.
HL63887, HL84207,
AHA13POST14410020, K99HL166776
Abstract MP09: Vascular Rho-kinase Activation by Inflammatory Signaling Mediates Blood Pressure Increase in Overweight
Authors: Aoqui Cristiane Aoqui , Chmielewski Stefan , Heemann Uwe , Baumann Marcus
Published: July 2021

Background:
Overweight is associated with a high prevalence of hypertension. The mechanisms linking overweight to blood pressure increase remain unclear. We hypothesized that vascular Rho-kinase activation contributes to blood pressure increase in overweight by involving TNF-α and TLR4.

Methods:
C57/BL6 mice fed a high-fat diet for 2 weeks were used to induce overweight associated blood pressure increase. Additional treatment in overweight and normal weight mice contained Rho-kinase inhibitors (fasudil and pravastatin; n=7/all groups), etanercept and TNFR1 and TLR4 null-mice. Microvascular studies were performed in a wire myograph and arterial blood pressure measured with a carotid catheter. Rho-kinase activity was determined in small mesenteric arteries of all groups. Inflammatory ligands such as TNF-alpha and free fatty acids were determined. Effects of TNF-alpha and TLR4 ligand LPS and palmitic acid on Rho-kinase activity and were determined ex vivo in mesenteric arteries and in in vivo.

Results:
Overweight mice had higher blood pressure (Delta: 9±2 mmHg) and vasoconstriction as normal weight mice. Small mesenteric arteries of overweight mice had a 50% higher Rho-kinase activity as normal weight mice. Ex vivo treatment with the Rho-kinase inhibitor Y-27632 reversed vasoconstriction of mesenteric arteries of overweight mice to constriction level in normal weight mice. In vivo treatment with the Rho-kinase inhibitor fasudil or pravastatin along with high-fat diet abolished the overweight associated blood pressure increase and enhanced vasoconstriction. TNF-alpha and TLR4 ligand free fatty acid were enhanced in overweight mice. TNF-alpha and TLR4 ligand LPS and palmitic acid increased Rho-kinase activity in mesenteric arteries. Use of etenercept, TNFR1 and TLR4 null-mice in the overweight model prevented blood pressure increase, vasoconstriction and Rho-kinase activation. All described effects were independent of adiposity.

Conclusion:
These results indicated that in diet-induced overweight vascular Rho-kinase activation is a key element of increased blood pressure and vasoconstriction. Potential activator of Rho-kinase are mediated by inflammatory factors including TLR4 ligands and TNF-alpha.
Abstract 002: Dynamic T Cell-Antigen Presenting Cell Interactions and Direct T Cell Activation Within the Vascular Wall During Hypertension
Authors: Wei Zihui , Wang Anna , Spizzo Iresha , Hickey Michael J , Widdop Robert E , Vinh Antony
Published: July 2021

T cells are now known to be vital to the development of experimental hypertension. Hypertension is associated with significant accumulation of T cells into the perivascular fat surrounding the aorta and renal vasculature. While a hypertension-specific neoantigen has been implicated in T cell activation, it is not known whether vascular-infiltrating T cells recognize and are locally activated by an antigen within the vessel wall. We developed live-cell imaging of explanted aortas to identify whether cognate antigens are presented to T cells within the vessel wall of hypertensive mice, evidenced by slower T cell velocities and a greater number of T cells interacting with antigen presenting cells (APCs). Splenic T cells were isolated from normotensive vehicle-treated (nT cells) and hypertensive angiotensin II (Ang II)-infused (0.7mg/kg/day; 14 days; hT cells) C57BL6/J mice. Following anti-CD3/CD28 stimulation (48 hours), cells were fluorescently labelled and co-incubated simultaneously (16 hours) with explanted aorta from normotensive or hypertensive CD11c-YFP mice, where APCs are fluorescently labelled. In CD11c-YFP mouse aorta alone, we detected a ~2-fold increase in CCR5 ligand (CCL3, CCL4 and CCL5) secretion from hypertensive mouse aorta compared to vehicle-treated mouse aorta (*P<0.05; n = 4). Using 2-photon microscopy, we observed a greater number (~2-fold) of hT cells compared to nT cells within Ang II-infused mouse aorta (390 ± 113 Vs 198 ± 49). Importantly, time-lapse recordings of hypertensive mouse aorta revealed hT cells exhibited significantly slower velocity (hT cells 2.0 μm/min Vs nT cells 4.8 μm/min; **P<0.01, n=8-12), and a greater proportion of interactions with APCs (hT cells 4.4 ± 1.1 Vs nT cells 0.8 ± 0.4%). Moreover, activation of local vascular T cells by incubating hypertensive aorta with anti-CD3/CD28 antibodies (16 hours) augmented Ang II-induced endothelial dysfunction (67.5 ± 2.0 Vs Ang II alone 54.5 ± 3.7% maximal relaxation). We have the first evidence that vascular-infiltrating T cells are presented with cognate antigens by APCs within the vessel wall during hypertension; direct activation of these T cell infiltrates further impairs endothelial function, which may promote the development of hypertension.
Abstract 005: Axl Controls Survival of the CD4+ T Lymphocytes in Salt-dependent Hypertension
Authors: Korshunov Vyacheslav A , Hughson Angie , Morrell Craig N , Fowell Deborah J , Batchu Sri N
Published: July 2021

Introduction:
Axl, a receptor tyrosine kinase, is required for vascular and immune cell survival. We sought to investigate the effects of Axl on T lymphocyte survival during deoxycorticosterone acetate (DOCA)-salt hypertension in mice.

Methods and Results:
We found significant reduction in systolic blood pressure (BP) after 5-6 weeks of DOCA-salt in RAG1-/- mice after adoptive transfer of CD4+ T cells from Axl knockout (Axl-/- →RAG1-/-) compared to transferred CD4+ T cells from wild type (Axl+/+ →RAG1-/-) mice. Media area of the mesenteric artery was significantly lower in Axl-/- →RAG1-/- (4.2±0.7x10
3
m
2
) vs. Axl+/+ →RAG1-/- (6.0±0.9x10
3
m
2
) or Axl+/+ (6.8±0.6x10
3
m
2
) mice. There was significant decrease in interferon gamma production by the T cells from Axl-/- (396±23 ng/mL) compared to Axl+/+ (512±42 ng/mL) after T
h
1-priming. The number of carboxyfluorescein succinimidyl ester-positive cells in 6
th
division was dramatically declined in Axl-/- (~0.3%) vs. Axl+/+ (~1.8%) in culture. Accordingly, we found lower number of lymphocytes in blood from Axl-/- (4.5±0.7x10
9
) compared to Axl+/+ (7.8±0.7x10
9
) mice. Blood leukocyte apoptosis was 2.5-fold higher in Axl-/- mice. We next investigated repopulation capacities of the hematopoietic cells from Axl-/- vs. Axl+/+ mice. There was significant decrease in Axl-/- CD3+ T cells (21±3 %) than Axl+/+ (49±3 %) in spleen after 8 weeks of competitive repopulation of bone marrow-derived cells. However, we found even greater reduction of Axl-/- T lymphocytes (15±1 %) vs. Axl+/+ T lymphocytes (52±6 %) in peripheral blood after 8 weeks of competitive repopulation. Finally, percentage of apoptotic cells was the greatest in the media (20±7 %) and adventitia (13±5 %) from Axl-/- →RAG1-/- mice compared to vascular apoptosis (6-14 % in media; and 6-9 % in adventitia) in other groups after 6 weeks of DOCA-salt.

Conclusions:
Our data suggest that Axl-dependent survival of the T lymphocytes is crucial for the late increase in BP in DOCA-salt hypertension.
Abstract 015: Increased 20-HETE Levels Contribute to Impaired Glucose Metabolism and Type 2 Diabetes in Cyp4a14 Knockout Mice Fed on High Fat Diet.
Authors: Pandey Varunkumar G , Bellner Lars , Garcia Victor , Schragenheim Joseph , Cohen Andrew , Falck John , Rocic Petra , Capdevila Jorge , Abraham Nader G , Schwartzman Michal
Published: July 2021

20-HETE (20-Hydroxyeicosatetraenoic acid) is a cytochrome P450 ω-hydroxylase metabolite of arachidonic acid that promotes endothelial dysfunction, microvascular remodeling and hypertension. Previous studies have shown that urinary 20-HETE levels correlate with BMI and plasma insulin levels. However, there is no direct evidence for the role of 20-HETE in the regulation of glucose metabolism, obesity and type 2 diabetes mellitus. In this study we examined the effect of 20-SOLA (2,5,8,11,14,17-hexaoxanonadecan-19-yl-20-hydroxyeicosa-6(Z),15(Z)-dienoate), a water-soluble 20-HETE antagonist, on blood pressure, weight gain and blood glucose in Cyp4a14 knockout (Cyp4a14-/-) mice fed high-fat diet (HFD). The Cyp4a14-/- male mice exhibit high vascular 20-HETE levels and display 20-HETE-dependent hypertension. There was no difference in weight gain and fasting blood glucose between Cyp4a14-/- and wild type (WT) on regular chow. When subjected to HFD for 15 weeks, a significant increase in weight was observed in Cyp4a14-/- as compared to WT mice (56.5±3.45 vs. 30.2±0.7g, p<0.05). Administration of 20-SOLA (10mg/kg/day in drinking water) significantly attenuated the weight gain (28.7±1.47g, p<0.05) and normalized blood pressure in Cyp4a14-/- mice on HFD (116±0.3 vs. 172.7±4.6mmHg, p<0.05). HFD fed Cyp4a14-/- mice exhibited hyperglycemia as opposed to normal glucose levels in WT on a HFD (154±1.9 vs. 96.3±3.0 mg/dL, p<0.05). 20-SOLA prevented the HFD-induced hyperglycemia in Cyp4a14-/- mice (91±8mg/dL, p<0.05). Plasma insulin levels were markedly high in Cyp4a14-/- mice vs. WT on HFD (2.66±0.7 vs. 0.58±0.18ng/mL, p<0.05); corrected by the treatment with 20-SOLA (0.69±0.09 ng/mL, p<0.05). Importantly, glucose and insulin tolerance tests showed impaired glucose homeostasis and insulin resistance in Cyp4a14-/- mice on HFD; ameliorated by treatment with 20-SOLA. This novel finding that blockade of 20-HETE actions by 20-SOLA prevents HFD-induced obesity and restores glucose homeostasis in Cyp4a14-/- mice suggests that 20-HETE contributes to obesity, hyperglycemia and insulin resistance in HFD induced metabolic disorder. The molecular mechanisms underlying 20-HETE mediated metabolic dysfunction are being currently explored.
Abstract 022: A Short Open Reading Frame in the Angiotensin Type 1a Receptor 5' Leader Sequence Increases the Rate of Receptor Internalization
Authors: Kadam Parnika , Mueller Susette , Ji Hong , Sandberg Kathryn
Published: July 2021

Background:
We recently found that a seven amino acid peptide (PEP7) encoded within a short open reading frame (sORF) in exon 2 of the 5’ leader sequence (5'LS) of the angiotensin type 1a receptor (AT1aR) mRNA inhibits AT1aR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) without having any effect on the AT1aR-inositol trisphosphate protein kinase C pathway. To investigate the mechanism by which PEP7 selectively inhibits the AT1aR-Erk1/2 signaling cascade, the start codon of the sORF was mutated at adenine -108 (A-108 to T-108) to create E1,2(-108T),3-AT1aR followed by cloning the E1,2,3-AT1aR and the mutant E1,2(-108T),3-AT1aR into the pEGFP-N2 plasmid.

Methods:
Human embryonic kidney 293 (HEK-293) cells were transfected with E1,2,3-AT1aR (intact sORF construct) or E1,2(-108T),3-AT1aR (disrupted sORF construct) by lipofectamine. Forty eight to seventy two hours later, live cell time lapse images were collected before and after treatment with Ang II (100 nM) using a TE300 Spinning disk laser scanning confocal microscope. Image analysis was performed using Velocity software.

Results:
Within 5 min of Ang II stimulation, punctae formed and moved throughout the cell membrane in cells transfected with both EGFP tagged receptors; however, even though the punctae represent the localized accumulation of identical AT1aR proteins, there were distinct differences in the intensity and time course of punctae. The rate of vesicle formation after Ang II treatment was markedly decreased by disrupting the PEP7 sORF [t1/2 (s): E1,2,3-AT1aR, 203s (N=21) vs E1,2(-108T),3-AT1aR, 328s (N=14); p<0.0001].

Conclusion:
The PEP7 sORF facilitates Ang II-induced AT1R internalization. These findings suggest that we have uncovered a new mechanism governing agonist-induced AT1aR cellular trafficking that could have implications not only for regulation of AT1aR signaling cascades but also for other trafficking proteins that contain an upstream sORF within their 5'LS.
Abstract 023: Collecting Duct (Pro)Renin Receptor Mediates Angiotensin II-induced Hypertension
Authors: Wang Fei , Peng Kexin , Lu Xiaohan , Yang Kevin , Liu Mi , Nau Adam , Chen Ren , Kohan Donald E , Feng Yumei , Ichihara Atsuhiro , Reese Van , Richardson Russell S , Zhou Shu-Feng , Yang Tian
Published: July 2021

Within the kidney, (pro)renin receptor (PRR) is predominantly expressed in the intercalated cells (IC) of collecting duct (CD) where its expression is induced by angiotensin II (AngII). Here we examined the function of PRR in the CD by analyzing mice with CD-specific deletion of PRR (CD PRR KO) using AQP2-Cre which has recently been shown to target both IC and principal cells (PC). Radiotelemetry demonstrated that the null mice were largely resistant to AngII-induced hypertension (MAP on day 7: Floxed/AngII 137.4 ± 3.5 vs. KO/AngII 121.2 ± 1.1 mmHg, p<0.05, n=4), accompanied with reduced urinary soluble PRR (sPRR) and aldosterone levels. Electrophysiology analysis demonstrated that within minutes activation of PRR by 10 nM prorenin induced a transient increase in amiloride-sensitive Na+ transport in cultured mpkCCD cells (Ieq: 1.85 ± 0.17 vs. 1.30 ± 0.06 μA/cm2, p<0.05). Interestingly, this was followed by a second phase of ENaC activation after 6 h, which reached the plateau activation at 24 h, accompanied with increased aldosterone release as assessed by ELISA (14.41 ± 0.92 vs. 5.45 ± 0.28 pg/ ml/μg protein, p<0.05). The chronic but not acute phase of ENaC activation was abolished by eplerenone. Both phases of ENaC activation depended on Nox4-derived reactive oxygen species (ROS). Immunostaining using an antibody against sPRR (the N terminus) showed exclusive labeling in the principle cells (PC) whereas the labeling with the C-terminal antibody was exclusively found in IC. A recombinant histidine-tagged sPRR, termed sPRR-His, in the nanomolar range induced a similar dual effect on ENaC activation as prorenin. Intravenous infusion of sPRR-His in CD PRR KO mice for 5 days completely restored the hypertensive response to AngII (MAP: 135.5 ± 7.5 vs. 116.7 ± 5.7 mmHg, p<0.05). We conclude that: 1) CD PRR mediates AngII-induced hypertension; 2) PRR activation in the CD leads to increased ENaC activity acutely through the direct action of ROS and chronically through local generation of aldosterone; 3) sPRR derived from IC may act in a paracrine fashion to stimulate Na+ transport in PC.
Abstract 033: Neuronal Knock-out of AT1 Receptor Attenuates the Development of Doca-salt-induced Hypertension
Authors: Xu Jiaxi , Sriramula Srinivas , Lazartigues Eric
Published: July 2021

We previously reported that neurogenic hypertension is associated with an increase in A Disintegrin And Metalloprotease 17 (ADAM17) activity and a reduction of Angiotensin Converting Enzyme 2 (ACE2) activity in the hypothalamus. In addition, we showed that silencing ADAM17 or blocking Angiotensin (Ang)-II type 1 receptors (AT1R) in the central nervous system (CNS) prevented DOCA-salt hypertension, confirming the pivotal role of AT1R and ADAM17 in neurogenic hypertension. However, the interaction between AT1R, ADAM17, and ACE2 is still unclear. Since ADAM17 is known to be expressed in multiple cell types and can be activated by various receptors, we tested the hypothesis that neuronal AT1R are necessary for ADAM17-mediated ACE2 shedding in neurogenic hypertension. Male neuronal AT1R knockout (AT1R floxed crossed with Nefh-cre recombinase mice, 12-16 week-old, n=5) and littermate mice (n=8) were implanted with telemetry probes for continuous recording of blood pressure (BP) and heart rate (HR). Following DOCA-salt treatment, both strains developed hypertension, however mean arterial pressure (MAP; 123.9 ±6.4 vs. 138.4 ±4.3 mmHg) and HR (483.9 ±24.1 vs. 520.3 ±22.5 bmp) were significantly lower in neuronal AT1R knockout mice after 2 weeks of treatment, compared to controls. Western blot and enzyme activity assays from the hypothalamus of these DOCA-salt-treated mice revealed that the expression of pro-ADAM17 (inactive form) was significantly increased (+17.0 ±5.3%, P<0.05), while the activity of ADAM17 was decreased (-37.4 ±10.5%) in neuronal AT1R knockout animals. Concomitant to the down-regulation of ADAM17, both the expression and activity of ACE2 were found to be significantly (P<0.05) up-regulated in the hypothalamus of neuronal AT1R knockout mice, by +20.7 ±8.5% and +32.3 ±10.1%, respectively. These results suggest that activation of neuronal AT1R is responsible for ADAM17-mediated ACE2 shedding and the maintenance of neurogenic hypertension.
Abstract 034: Opposite Effects of Hypertension and Training on Blood Brain Barrier Integrity in Autonomic Areas of the Spontaneously Hypertensive Rat
Authors: Michelini Lisete C , Jordao Maria T , Ceroni Alexandre , Buttler Leila
Published: July 2021

It is well known that chronic hypertensive rats exhibit deficient blood brain barrier (BBB). We evaluate age-induced progression of BBB lesion in autonomic areas of the SHR and the possible effect of aerobic training on BBB integrity. SHR aged 1, 3 and 5 month were chronically cannulated for hemodynamic recordings in the conscious state (femoral), followed by anesthesia and dextrans’ infusion (FITC-10kDA + RHO-70kDA, carotid artery). Twenty minutes later rats were sacrificed, brains were removed, post-fixed and cryoprotected. BBB permeability was evaluated in sequential 30 μm slices of the hypothalamic paraventricular nucleus (PVN), nucleus tractus solitarii (NTS) and rostroventrolateral medulla (RVLM) by the capability of FITC10 to leak into the brain parenchyma (in % area/area of interest, fluorescent microscope, ImageJ analysis). Other 3-months old SHR were submitted to treadmill training (T=55% of maximum capacity, 1h/day, 5 d/week) or kept sedentary for 8 weeks. Age-matched WKY served as control. Although SHR aged 1 month were normotensive and exhibited no BBB leakage (0.02±0.01 to 0.15±0.03% in all areas, values similar to WKY), leakage augmented sharply with the establishment of hypertension (average of 8.1±0.7% in SHR-3mo, 9.3±1.2% in SHR-5mo). In the WKY, there was only a small age-induced increase in BBB leakage (average of 0.63±0.05% in PVN, NTS and RVLM). Interestingly T promptly reduced dye leakage in the 3 autonomic areas of the SHR (~1.0±0.2% from 2 up to 8 weeks of training) without changing the leakage in PVN, NTS and RVLM of WKY rats and within the hypoglossus nucleus of the SHR (a non-autonomic area). T-induced improvement of BBB integrity in autonomic areas of the SHR were accompanied by significant reductions of HR (-10%) and MAP (-13%), increased HR variability (+2.1-fold), decreased pressure variability (-49%) and increased spontaneous baroreflex sensitivity (+2.3-fold). Data show that BBB lesion in SHR is caused by the establishment of hypertension and that T improves perfusion of autonomic brain areas in hypertensive individuals by preserving BBB integrity. This adaptive response is crucial for a near normal neuronal activity, thus normalizing autonomic control of the circulation even in the presence of hypertension.
Abstract 047: Genetics and Environmental Influences on Ambulatory Blood Pressure in Rats Compared With Circadian Blood Pressure Variability in Human
Authors: Lee Mary S , Lee John S , Lee Jong Y , Azar Silvia H , Halberg Franz
Published: July 2021

Genetic susceptibility is an important factor in raising blood pressure (BP). Daily (circadian) rhythm characteristics are considered essential parameters for recognizing and treating increased risks in BP. To examine BP in genetics with environmental modifications, one-cell homozygous embryos were transferred into spontaneously hypertensive (SHR, pup:shr) or normotensive (WKY, pup:wky) rats' oviducts (embryos: s, w; oviduct-uterine: S, W) in a reciprocal fashion. Pups were cross-suckled at birth (nurses S, W) and weaned to normal diets at day-21. At Day-120, telemetered BPs were monitored for 5 consecutive days every 4 min and analyzed by the method of ANOVA. Ambulatory BP in 20 adolescents and adults were monitored automatically around the clock at 30-min to hourly intervals and analyzed by the least square rhythmometry method.

As expected, shr BPs were markedly reduced when they were transplanted to the W-uterine and/or the W-lactation milieu (sSS vs. sSW, sWS and sWW: 197 vs.178, 147 and 178 mm Hg). BP in wky was significantly altered only in the wSW group (wWW vs, wSW: 127 vs.131 mm Hg). All subjects showed significant circadian fluctuations with a peak in the late afternoon hours in most human subjects and rats as a nocturnal animal mostly close to midnight hours, while shr with W-uterine (sWS) a bit delayed peak hour (00:45) and with combined W-uterine/W-nursing (sWW) a bit earlier peak hour (20:12). Circadian double amplitudes (2A) in the human subjects varied from 8 to 26 mm Hg with higher 2A in elder adults, and 3-8 mm Hg in rats with significantly higher fluctuations in shr groups as compared to that of wWW (7.5±0.7 for sSS, 8.3±0.6 for sSW vs. 4.7±0.3 mm Hg for wWW).

The hypertensive-prone shr strain showed significantly lowered BPs in a normotensive WKY uterine environment and/or by WKY nursing mothers, indicating that environment influences can strongly modify genetic factors, yet the lowered shr MESORs by the WKY environments remained above the MESORs encountered in wky donors. Chronomes broader than circadian should be considered in interpreting BP responses as a gauge of vascular disease status.
Abstract 052: Placental Growth Factor (PlGF) Reduces Blood Pressure and Improves Endothelin Type B Receptor (ETBR)-Mediated Microvascular Dilation in Hypertensive Pregnant Rats
Authors: Mazzuca Marc Q , Ren Zongli , Lin Chen , Possomato-Vieira Jose S , Zhu Minglin , Khalil Raouf A
Published: July 2021

Preeclampsia is a pregnancy-related hypertensive disorder (HTN-Preg) with an imbalance between anti-angiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and angiogenic PlGF, but the vascular targets involved are unclear. We have shown downregulation of endothelial ET
B
R in Preg rats with reduced uterine perfusion pressure (RUPP), and studies have shown increased plasma sFlt-1 in RUPP rats. We tested if raising PIGF/sFlt-1 ratio by infusing PIGF (10 μg/kg/day) in RUPP rats would improve BP and microvascular ET
B
R signaling, and vice versa, if lowering PIGF/sFlt-1 ratio by infusing sFlt-1 (10 μg/kg/day) in Preg rats increases BP and reduces ET
B
R signaling. On day 19, BP was recorded and mesenteric microvessels were isolated for measurement of diameter and [Ca
2+
]
i
(fura-2 340/380 ratio). BP was in PlGF-RUPP 105±2 < RUPP 126±1 and in sFlt-Preg 125±4 > Norm-Preg 97±5 mmHg. ET-1 vasoconstriction was in PlGF-RUPP 62.6±3.0 < RUPP 83.4±5.3 and in sFlt-Preg 76.1±4.7 > Norm-Preg 52.1±3.2%. ET-1 caused parallel increases in microvascular [Ca
2+
]
i
that was in PlGF-RUPP 0.87±0.02 < RUPP 0.92±0.01 and in sFlt-Preg 0.93±0.02 > Norm-Preg 0.85±0.01. Endothelium removal or microvessel treatment with ET
B
R antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca
2+
]
i
in Norm-Preg and PlGF-RUPP, but not RUPP or sFlt-Preg. The ET
B
R agonists sarafotoxin 6c (S6c) and IRL-1620 caused relaxation that was in PlGF-RUPP 42.9±10.8, 38.0±11.2% > RUPP 4.7±3.4, 7.5±2.3% and in sFlt-Preg 3.1±1.0, 5.4±1.6% < Norm-Preg 29.9±7.8, 28.0±9.1%. L-NAME partially reduced ACh- and ET
B
R-induced relaxation in Norm-Preg, PlGF-RUPP, but not RUPP or sFlt-Preg, suggesting that PlGF improves the decreased NO-dependent and ET
B
R-mediated vasorelaxation in HTN-Preg. Basal, ACh-, S6c-, and IRL-1620-induced nitrate/nitrite production was enhanced in mesenteric arteries of PIGF-RUPP and Norm-Preg vs. RUPP rats. Western blots and immunohistochemistry revealed greater levels of endothelial ET
B
R in PlGF-RUPP and Norm-Preg vs. RUPP and sFlt-Preg. Thus improving PlGF/sFlt-1 balance reduces BP and ET-1 vasoconstriction, and enhances ET
B
R-mediated NO-dependent vasodilation in RUPP rats, and could be a new approach in the management of HTN-Preg.