bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 020: Physiological Significance of Angiotensin AT
1A
Receptors in Vasopressin-producing Cells of the Supraoptic Nucleus
Authors: Linggonegoro Danny W , Sandgren Jeremy A , Claflin Kristin E , Perschbacher Katherine J , Ni Jonathan , Pearson Nicole A , Pierce Gary L , Santillan Mark K , Grobe Justin L
Published: June 2021

Low-renin and salt-sensitive forms of hypertension are characterized by elevated activity of the brain renin-angiotensin system and secretion of arginine vasopressin (AVP). While angiotensin in the brain is a known stimulant of AVP secretion through its AT
1
receptor, the localization of relevant AT
1
receptors remains unclear. We tested whether AT
1A
receptors localized to AVP-producing cells are important for AVP secretion. To examine AVP and AT
1A
co-localization, mice expressing Cre-recombinase via the AVP gene (AVP-Cre) were bred with mice expressing a conditional red fluorescent ROSA-stop
flox
-tdTomato construct and GFP via an AT
1A
BAC transgene. Dual-fluorescent cells were detected in supraoptic nuclei (SON) but not paraventricular nuclei. Mice lacking AT
1A
specifically in AVP-producing cells (AT
1A
AVP-KO
) were then generated by breeding AVP-Cre mice with mice harboring a conditional endogenous AT
1A
gene. AT
1A
AVP-KO
mice exhibited normal serum (littermate n=13, 353±69 vs AT
1A
AVP-KO
n=7, 207±37 pg/mL, p=NS) and urine (n=26, 145±33 vs n=11, 170±54 pg/mL, p=NS) copeptin (the stable C-terminal fragment of AVP) as well as hematocrit (n=14, 46.3±0.7 vs n=7, 47.5±1.3 %, p=NS) despite increased serum osmolality (n=33, 324±1.3 vs n=19, 330±1.6 mOsm/kg, p<0.01), supporting a role for AT
1A
in AVP-producing cells in modulating the osmotic control of AVP release. Systolic blood pressure (SBP) (n=18, 109±1.3 vs n=5, 107±1.2 mmHg), urine volume (n=27, 1.1±0.1 vs n=12, 0.9±0.2 mL/d), and fluid intake (n=27, 4.0±0.2 vs n=12, 3.9±0.2 mL/d) were all normal (p=NS) in AT
1A
AVP-KO
mice. Two-bottle choice between water and escalating concentrations of NaCl uncovered minor alterations in sodium intake behavior. Serum osmolality (n=22, 336±2 vs n=9, 333±3 mOsm/kg), SBP (n=23, +10.4±2.1 vs n=8, +12.9±2.0 mmHg), urine output (n=23, +12.7±0.8 vs n=9, +12.7±1.5 g/day), and fluid intake (n=23, +16.2±1.3 vs n=9, +14.8±2.5 mL/day) all increased normally (p=NS) in response to deoxycorticosterone acetate (DOCA)-salt treatment. Collectively these data support a role for AT
1A
receptors, localized specifically to AVP-expressing cells of the SON, in the normal osmotic control of AVP secretion.
Abstract 027: Participation of Cardiac Thyrotroping Releasing Hormone (cTRH) in the Doxorrubicin (D) Induced Cardiotoxicityin in Mice
Authors: Peres Diaz Ludmila S , Schuman Mariano L , Aisicovich Maia , Landa Maria S , García Silvia I
Published: June 2021

We have demonstrated TRH hyperactivity in the hypertrophied Left ventricle (LV) of SHR. Its specific inhibition attenuates hypertrophy development in spite of the significant higher pressure observed (Schuman M, Hypertension 2011). LV-TRH over-expression induces several features of the hypertrophied heart, including increases in the apoptotic index Bax/Bcl2, and the activated caspase 3 observed by immunohystochemistry (Schuman M, AJPH&C 2014). In addition, we have found that TRH expression was induced by D in primary cultures of cardiac cells. Based on these results we hypothesized that cTRH could participate in the D cardiotoxicity effects. Indeed, we used C57 adult males (n=10) with a single D or saline injection (200uL,10 mg/kg ip) which previously (24 h, under anesthesia) received an intracardiac injection of a specific TRH-siRNA to inhibit LV-TRH expression or scrambled Con-siRNA. Mice were sacrificed 2,4 and 7 days post D injection and body weight was measured. Genes expression was measured by real time PCR and protein by immunohystochemistry (ANOVA and Tukey test). Body weight showed a mild but not significant decrease in D treated animals. D significantly increased TRH gene expression and TRH protein content reaching the maximum at 7 days post injection (2d: 145%, 4d:190% and 7d: 250%) (p& 0.05), which were not observed in the groups with D+TRH-siRNA indicating the effectiveness of the specific TRH inhibition. Also at this time TRH inhibition attenuates (p&0.05) D-induced increase in the apoptotic index Bax/Bcl2 and the augmented activated caspase 3 content pointing out the participation of the cardiac TRH in the D-induced apoptosis. Similar results were observed with hypertrophic and fibrotic markers gene expression (BNP, BMHC and col III) which showed a significant increase (p& 0.05) only in the groups with D and the intact TRH system (D+Con-siRNA). Fibrosis results were confirmed by Sirius Red and Masson techniques.

On the whole, we demonstrated for the first time that LV-TRH system is required for both, Doxorrubicin induction of apoptosis and consecutively hypertrophy and fibrosis in the mouse heart. Even more, we found that cTRH inhibition attenuates doxorrubicin induced damage suggesting a novel mechanisms in the cardiotoxicity injury.
Abstract 031: Placental Ischemia Causes Mitochondrial Dysfunction in Reduced Uterine Perfusion Pressure Rats
Authors: Vaka Venkata Ramana , Cunningham Mark W , Ibrahim Tarek , Amaral Lorena M , LaMarca Babbette
Published: June 2021

Introduction:
Placental ischemia is believed to be the initial event in the development of preeclampsia (PE). PE is characterized by new onset hypertension and is associated with reduced fetal weight and placental oxidative stress. Mitochondrial dysfunction is the major cause of reactive oxygen species (ROS) generation. We hypothesize that the placental ischemia causes mitochondrial dysfunction in the reduced uterine perfusion pressure (RUPP) rat model of PE. Thus, the purpose of this study is to examine the effect of placental ischemia on mitochondrial function.

Methods:
Female Sprague Dawley rats were dived into two groups; normal pregnant (NP) and RUPP rats. On gestational day (GD) 14, RUPP surgery was performed, GD18 carotid catheters were inserted, and GD19 conscious blood pressure (MAP) was measured. GD 19 placentas were collected and mitochondria were isolated for respiration measurements. Respiration measurements included: basal state (isolated mitochondria with no substrates), state 2 (glutamate and malate as complex I substrates), state 3 (ADP stimulated), leak state (oligomycin induced ATP synthase inhibition), and maximal state (FCCP stimulated uncoupled). Cytochrome C oxidase (complex IV) activity was also measured in isolated mitochondria.

Results:
MAP was elevated in RUPP (n=16) compared to NP rats (n=14) (119±2 vs. 100±2 mmHg, p<0.05). State 3 (149±8 vs 220±12 pmol/sec/mg, p<0.05) and maximal (123±8 vs 159±5 pmol/sec/mg, p<0.05) respiration rates were significantly reduced in RUPP (n=5) vs NP (n=7) rats. However, basal, state 2, and state 4 respiratory rates were not different between RUPP vs NP. Respiratory control ratio (state 3/ state 4) was significantly reduced in RUPP (n=5) vs NP (n=7) (7±1 vs 10±1, p<0.05). Complex IV activity was reduced in RUPP (n=5) vs. NP (n=6) rats (508±14 vs 644±17 nmol e-/min/mg, P<0.05).

Conclusion:
Reduced mitochondrial respiration and cytochrome c oxidase activity, indicators of mitochondrial dysfunction, occur in response to placental ischemia during pregnancy. Thus, suggesting that mitochondrial dysfunction may contribute to the pathophysiology of oxidative stress and hypertension during PE.
Abstract 035: Differential Effects of Bardet-biedl Syndrome 1 Gene Deletion From Agouti-related Peptide Neurons
Authors: Guo Deng F , Reho John J , Morgan Donald A , Rahmouni Kamal
Published: June 2021

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive human disorder associated with several clinical features including obesity and hypertension. We previously demonstrated that
Bbs1
gene deletion from the central nervous system caused obesity and hypertension in mice highlighting the importance of neuronal BBS proteins for energy homeostasis and cardiovascular regulation. Here, we investigated the role of Bbs1 gene in the orexigenic agouti-related peptide (AgRP)
neurons.
We crossed Bbs1
fl/fl
mice with AgRP
Cre
mice and confirmed Cre recombinase activity in AgRP neurons using td-Tomato reporter mice. Interestingly, female AgRP
Cre
/Bbs1
fl/fl
mice developed profound obesity as indicated by the increased body weight (46.8±2.0 g vs control littermates, 35.5±1.9 g at 25 weeks of age). In contrast, body weight of male AgRP
Cre
/Bbs1
fl/fl
mice (48.5±1.2 g) was similar to the controls (46.7±1.2 g). There was no change in the expression of hypothalamic genes regulating food intake (AgRP, proopiomelanocortin and neuropeptide Y) in AgRP
Cre
/Bbs1
fl/fl
mice relative to controls. Next, we assessed the consequence on arterial pressure (AP) and sympathetic nerve activity (SNA) of ablating the
Bbs1
gene from AgRP neurons. Interestingly, deletion of the
Bbs1
gene in AgRP neurons did not recapitulate the hypertension phenotype of BBS as indicated by the lack of difference in mean AP (107±5.0 vs 106±5.2 mmHg in controls) and heart rate (597±23.5 vs 589±16.4 in control). However, conscious renal SNA was significantly higher in AgRP
Cre
/Bbs1
fl/fl
mice relative to controls (114±10 vs 78±6 spikes/sec, p<0.05). In addition, the depressor effect of ganglionic blockade (hexamethonium) was higher in AgRP
Cre
/Bbs1
fl/fl
mice (-64.4±10 vs -55.8±8.3 mmHg in control). Finally, we used wire myography to measure mesenteric artery vascular function and found impairment in acetylcholine induced relaxation in AgRP
Cre
/Bbs1
fl/fl
mice (Max. relaxation: 78 ± 6% vs 26 ± 9% in controls; p<0.05), but no changes in the relaxation evoked by sodium nitroprusside, suggesting endothelial, but not smooth muscle, dysfunction. These findings demonstrate that
Bbs1
gene in AgRP neurons is critical for energy homeostasis, renal sympathetic outflow and vascular endothelial function.
Abstract 040: Comparison of Effects of Reux en Y Gastric Bypass and Intra-abdominal Lipectomy on Cardiovascular Function in the Metabolic Syndrome
Authors: Soler Amanda , Hutcheson Brenda , Yang Jenny , Bradford Chastity , Zhang Frank , Gotlinger Katie , Schwartzman Michal L , Rocic Petra
Published: June 2021

Central (visceral) obesity is a key feature of the metabolic syndrome and an independent predictor of cardiovascular disease. Reux en Y gastric bypass (RnY) has been shown to offer protection against cardiovascular disease, but residual risk remains. It is also unknown whether the cardiovascular benefit is a consequence of a decrease in visceral (intra-abdominal) adipose tissue or of other factors. In this study, we compared the effects of RnY vs. removal of 90% of visceral adipose tissue (=5% body weight) by intra-abdominal lipectomy on cardiac function (echocardiography), macrovascular function (carotid artery stiffness) and microvascular function (coronary artery endothelium-dependent vasorelaxation) in a metabolic syndrome rat model (JCR:LA-cp, JCR). Cardiac output (CO) and ejection fraction (EF) were significantly decreased in JCR vs. normal (Sprague-Dawley, SD) rats (CO=50±5%, EF=45±2% of normal), and were significantly improved by both RnY and intra-abdominal lipectomy (CO=75±6%, EF=82±2% and CO=80±3%, EF=90±2% of normal, respectively). Likewise, acetylcholine-dependent coronary artery vasorelaxation was impaired in JCR rats (50±1% of normal), and was significantly improved by both RnY and intra-abdominal lipectomy (98±2% and 98±3% of normal, respectively). Carotid artery stiffness was significantly increased in JCR rats (~2 fold vs. SD), and was normalized by intra-abdominal lipectomy (to equal SD), but not by RnY (~2 fold vs. SD). Intra-abdominal lipectomy but not RnY also decreased cardiac and vascular elastin degradation in JCR rats (Lipectomy: ~50% (heart), ~75% (carotid); RnY: ~15% (heart), ~5% (carotid) vs. untreated JCR, respectively), concomitant with a decrease in matrix metalloproteinase 12 (MMP12), a major elastase, activation (~50% (heart), ~75% (carotid), ~87% (visceral fat), ~75% (circulating) vs. untreated JCR) and in 20-hydroxyeicosatetraeonic acid (20-HETE) levels (~4 (heart), ~7 (carotid), ~4 (visceral fat), ~4 (circulating) fold vs. untreated JCR). Thus, our data indicate that intra-abdominal adipose tissue itself is a source of factors that may be important negative regulators of micro- and macrovascular and cardiac function, but are not eliminated by RnY.
Abstract 048: Expression of a Hypertension-causing Mutation in Cullin 3 (CUL3Δ9) Specifically in Smooth Muscle Causes Vascular Dysfunction and Hypertension
Authors: Agbor Larry N , Wu Jing , Ibeawuchi Stella-Rita C , Hu Chunyan , Davis Deborah R , Keen Henry L , Quelle Frederick W , Sigmund Curt D
Published: June 2021

Pseudohypoaldosteronism type II (PHAII) patients with mutations in cullin 3 (CUL3) resulting in exon 9 deletion (CUL3Δ9), exhibit severe early onset hypertension correlated with impaired kidney function. However, the extra-renal mechanisms remain uninvestigated. We hypothesized that expression of CUL3Δ9 protein in smooth muscle in mice impairs endogenous wildtype CUL3 (CUL3-WT) function and causes vascular dysfunction and hypertension. We generated transgenic mice inducibly expressing CUL3Δ9 protein in smooth muscle (S-CUL3Δ9) and measured blood pressure (BP) by radiotelemetry. We assessed vascular responses in the cerebral basilar artery and aorta using a pressurized and a wire myograph, respectively. S-CUL3Δ9 mice exhibited reduced expression of endogenous CUL3WT protein compared to non-transgenic (NT) in aorta. Systolic BP was significantly increased in S-CUL3Δ9 mice (127±2 S-CUL3Δ9 vs 117±1 NT, p=0.02). Basilar artery from S-CUL3Δ9 mice exhibited significantly impaired vasorelaxation to acetylcholine (ACh) (at 100 μM: 15±4% S-CUL3Δ9 vs 65±5% NT, p<0.0001), and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μM: 59±2% S-CUL3Δ9 vs 90±5% NT, p<0.05). Vasocontraction to angiotensin II (Ang II), phenylephrine (PE) and to endothelin 1 (ET-1) were significantly elevated in S-CUL3Δ9 transgenic mice. Consistent with data from basilar artery, aorta from S-CUL3Δ9 transgenic mice exhibited impaired ACh-mediated relaxation (at 100 μM: 55±2% S-CUL3Δ9 vs 71±7% NT, p<0.0001). Total RhoA protein was significantly elevated in aorta of S-CUL3Δ9 transgenic mice (1.6±0.2 S-CUL3Δ9 vs 1.0±0.1 NT, P<0.05). Serotonin stimulation caused a significant increase in active RhoA in S-CUL3Δ9 aorta (1.83±0.04 S-CUL3Δ9 versus 1.52±0.06 NT, p=0.005). Preincubation with the Rho-kinase inhibitor (Y27632) restored endothelium-dependent relaxation in basilar artery and aorta of S-CUL3Δ9 mice. Ang II infusion via osmotic minipump (200 ng/kg/min) resulted in elevated BP response (Systolic BP: 147 ± 2 S-CUL3Δ9 versus 130 ± 5 NT, p=0.04) and increased aortic stiffening in S-CUL3Δ9 mice. We conclude that CUL3Δ9 acts in a dominant negative manner by interfering with CUL3-WT and contributes at least in part to hypertension via its effects on the vasculature.
Abstract 054: Mechanical Stretch on Endothelial Cells Promotes Monocyte Differentiation Into Immunogenic Dendritic Cells
Authors: Loperena Roxana , Chen Wei , Kirabo Annet , Harrison David G , Gomez Jose A
Published: June 2021

We have shown that dendritic cells (DCs) from hypertensive mice and monocytes in humans accumulate highly reactive isolevuloglandins (isoLGs) or isoketals that adduct to protein lysines and promote T cell activation, specifically. Monocytes that traverse the endothelium have three different fates: reemerge into circulation as an activated monocyte; differentiate into macrophages or into monocyte-derived DCs. We hypothesized that human endothelial cells exposed to hypertensive mechanical stretch will promote conversion of human monocytes into immunogenic DCs. We co-cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the HAEC monolayer to either normal cyclical stretch (5%) or hypertensive uniaxial cyclical stretch (10%) for 48 hours. We found that co-culture of monocytes with HAECs exposed to 10% mechanical stretch markedly increased monocyte mRNA expression of the Th17 polarizing cytokines IL-6, I-23A and IL-1β and the p22

phox

subunit of the NADPH oxidase compared to 5% stretch. HAECs exposed to 10% stretch promoted monocytes in culture to differentiate into DCs, as evidenced by the surface expression of DC-SIGN, CD83 and the co-stimulatory marker CD86. These monocytes also accumulated isoLG-modified proteins compared to controls (69.73 ± 5.802 vs 10.79 ± 1.854 respectively,
p
= 0.0001) as evidenced by intracellular staining and flow cytometry. We also co-cultured these monocytes with T cells from the same patient and examined proliferation of the latter using CFSE. Monocytes co-cultured with 10% stretched HAECs induced a 1,500-fold increase in CD4
+
T cell proliferation and a 1,300-fold increase in CD8
+
T cells proliferation. In addition, monocytes co-cultured with 10% stretched HAECs had a 2-fold increase in phosphorylated STAT3 expression compared to 5% stretch cultures. Conversely, monocytes-HAEC cultures exposed to 10% stretch and treated with STAT3 inhibitor, stattic, reduced their differentiation into DCs and prevented both CD4
+
and CD8
+
T cell proliferation. These data show that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote differentiation into activated DCs potentially via the STAT3 pathway.
Abstract 055: Benefit of Mineralocorticoid Receptor Antagonism in Acute Kidney Injury: Role of Smooth Muscle Rac1
Authors: Barrera-Chimal Jonatan , André-Grégoire Gwennan , Prince Sonia , Kolkhof Peter , Sauzeau Vincent , jaisser Frederic
Published: June 2021

Introduction:
Renal ischemia/reperfusion (IR) is a major cause of acute kidney injury (AKI). The benefit of novel non-steroidal MR antagonists such as finerenone in the IR context has not been evaluated and the mechanisms underlying the benefit of MR antagonism remain unclear.

Objectives:
To test the efficacy of finerenone in ischemic AKI and to evaluate the specific contribution of the MR expressed in endothelial or smooth muscle cell (SMC) in renal IR injury.

Methods:
We included 18 male C57/B6 mice that were divided in: sham, renal ischemia for 20 min and IR plus treatment with finerenone (10 mg/kg) by gavage once a day at -48, -24 and -1 h before IR. Alternatively, MR inactivation in endothelial cells (MR
endoKO
mice /Vecadh-cre) or in smooth muscle cells (MR
SMCKO
mice/SMA-cre) was induced in 3-month-old mice. Sham surgery or bilateral renal IR for 20 min was performed and mice were studied 24 h after reperfusion. Primary rat SMC cultures were used to assess the signaling pathways modulated by MR.

Results:
In C57/B6 WT, MR
fl/fl
and MR
endoKO
mice, IR induced kidney dysfunction and tubular injury. After IR, Finerenone-treated mice and the MR
SMCKO
mice presented normal renal function and a significant reduction of histological alterations, while MR
endoKO
mice were not protected. The benefit of finerenone and MR KO in SMC was associated with reduced oxidative stress-mediated lipid peroxidation as compared to MR
fl/fl
or WT mice. In aldosterone-stimulated rat SMC, we observed a 100% increase in hydrogen peroxide production and a 2-fold increase in Rac1 activity; MR and Rac1 antagonism blunted these effects. Moreover, mice deficient of Rac1 in SMC were also protected against ischemic AKI.

Conclusion:
Finerenone limits renal injury induced by IR. Moreover, genetic deletion of MR in SMC only has similar effects. This benefit was associated with reduced oxidative stress, by affecting oxidative stress production via SMC Rac1.
Abstract 057: Nox5 Induces Vascular Dysfunction and Arterial Remodelling Independently of Blood Pressure Elevation in Ang II-infused Nox5-expressing Mice
Authors: Montezano Augusto C , Harvey Adam P , Rios Francisco J , Dulak-Lis Maria , Beatie Wendy , McPherson Laura , Holterman Chet E , Kennedy Christopher R , Touyz Rhian M
Published: June 2021

Nox5 is a unique Ca
2+
-sensitive Nox isoform that is expressed in human vascular smooth muscle cells (VSMC). Although Nox5 has been implicated in diabetic nephropathy, its role in vascular function and development of hypertension remain unclear. Nox5 is not expressed in rodents, and accordingly we generated humanised Nox5 mice with Nox5 expressed in a VSMC-specific manner (Nox5SM22). Control (wild-type) and Nox5SM22 mice were infused with Ang II (600 ng/Kg/day). Blood pressure (BP) was assessed by tail-cuff. Vascular function and structure of resistance arteries were measured by myography. Ang II increased BP in WT (182.5±10 mmHg) and Nox5SM22 mice (173.1±5 mmHg) with no significant differences. Arteries from Nox5SM22 mice exhibited reduced endothelium-dependent relaxation versus WT controls (%ACh relaxation: 55.1±4 vs ctl: 81.6±7%). Fasudil (Rho kinase inhibitor)-induced relaxation was reduced in Nox5SM22 mice versus controls (%Fas: 111.3±11 vs ctl: 166.6±8%) (p<0.05). Ang II increased the maximal contraction to U46619 (thromboxane A2 mimetic) in WT (115.8±2 vs untreated: 101.4±2%) and Nox5SM22 (121.3±3 vs untreated: 99.1±2) (p<0.05) and induced endothelial dysfunction in all groups. Fasudil-induced relaxation was impaired by Ang II in WT (102.7±6 vs untreated: 166.6±8%, p<0.05) but not further impaired in Nox5SM22 mice (114.9±6 vs untreated: 111.3±11%). Ang II increased cross-sectional area (CSA) and lumen diameter; while in Nox5SM22 mice, Ang II increased wall thickness, wall-to-lumen ratio, CSA and decreased lumen diameter, with associated increased vascular stiffness. Our findings indicate that in mice expressing human Nox5 in VSMCs, endothelium-dependent relaxation is impaired, fasudil-mediated vasodilation is attenuated and vessels undergo exaggerated hypertrophic inward remodelling with increased stiffness; processes that occur independently of BP elevation. These data suggest an important role for Nox5 in Ang II-induced vascular dysfunction and remodeling, but not in the development of hypertension. Moreover, we identify Rho kinase as a putative target for Nox5-induced vascular injury. We provide novel insights into Nox5 vascular biology and demonstrate that vascular Nox5 actions are dissociated from BP effects.
Abstract 059: Kidney Specific DPP4 Knockdown Prevents Ang II/DOCA Salt Injury and Proteinuria
Authors: An Jianzhong , Liu Qiuer , Smith Cassandra , Nistala Ravi
Published: June 2021

Dipeptidyl peptidase 4 (DPP4) inhibition is widely used for glycemic control in type 2 diabetes mellitus (T2DM) subjects. The Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus - Thrombolysis in Myocardial Infarction 53 (SAVOR-TIMI 53) trial showed that DPP4 inhibition improves proteinuria in type 2 DM (T2DM) subjects with chronic kidney disease (CKD). Our lab and others have observed that kidney DPP4 activation is associated with proteinuria in conditions of RAAS activation including obesity. However, it is not clear if kidney specific DPP4 directly plays a role in kidney injury. To test our hypothesis, right nephrectomy (UNx) mice were subjected to 1) sham surgery 2) left renal vein injection with lentivirus carrying DPP4 shRNA (UNx+DPP4) 3) scramble shRNA (UNx+SCRM) and the animals were allowed to recover for 2-3 wks. A 4
th
group of non-UNx mice were added as additional controls. Next, angiotensin II (Ang II, 1000ng/kg/min) plus deoxycorticosterone (DOCA, 50mg) plus 0.9% NaCl (salt) or saline only was given for 2 wks. Ang II/DOCA salt caused heart hypertrophy in all groups when compared to saline infusion (p<0.05). DPP4 knockdown reduced albuminuria (UNx+Ang II/DOCA salt, 80.18±15.3 ug/mg Crt; UNx+DPP4+Ang II/DOCA salt, 25.76±19.3 ug/mg Crt; UNx+SCRM+Ang II/DOCA salt 100.1±17.6 ug/mg Crt, p<0.05). Flow cytometry analysis showed that DPP4 knockdown mitigated activation of CD4+ T cells by Ang II/DOCA salt as indicated by reduced surface expression of DPP4 on CD4+, CD44+CD127+ central memory and CD44+CD127- effector cells. In addition, DPP4 knockdown prevented Ang II/DOCA salt-mediated infiltration of Ly6ChiCD11bhiF4/80lo macrophages and decreased the Ly6C-CD11bloF4/80hi resident dendritic cells. DPP4 knockdown suppressed Ang II/DOCA salt mediated activation of pro-fibrotic markers, Col1A and CTGF. Lastly, DPP4 knockdown suppressed glomerulomegaly, mesangial expansion and interstitial inflammation on trichrome stain. In summary, RAAS activation is a prominent mechanism for kidney injury in T2DM and obese subjects. Kidney DPP4 is an important effector of RAAS mediated kidney inflammation, activation of pro-fibrotic markers and proteinuria, thereby implicating DPP4 in CKD initiation and progression.
Abstract 061: Cell Fate Changes During Tubular Damage and Regeneration in the Mouse Kidney
Authors: Nagalakshmi Vidya K , Li Minghong , Gomez R. A , Sequeira-Lopez Maria Luisa S
Published: June 2021

Tubular degeneration, loss of renal tubules and interstitial fibrosis due to kidney injury lead to chronic renal disease and hypertension. Using a partial unilateral ureteral obstruction (pUUO) model in neonatal mice, we analyzed the fate cell changes that occur during obstruction and during recovery following the release of UUO. We traced the fate of cells derived from the renal stroma, cap mesenchyme and ureteric bud epithelium using Foxd1-Cre, Six2-Cre and HoxB7-Cre mice respectively, crossed with double fluorescent reporter (mT/mG) mice. pUUO was performed 24-36h after birth (n=84). In a group of pups (n=37), the obstruction was released after seven days. Sham operated animals (n=35) were used as controls. Lineage tracing revealed that Foxd1-derived interstitial pericytes acquired α-smooth muscle actin expression and underwent significant expansion due to pUUO (fibrotic area 91.06+/-6.77 %). Release of obstruction resulted in complete resolution of fibrotic areas (0.00%; p<0.005). Further, loss of Six2-derived cells at the glomerular-tubular junction in pUUO kidneys resulted in the formation of atubular glomeruli (39%). Atubular glomeruli were not observed after release. In addition, a significant loss of HoxB7-derived collecting duct tubules was observed during pUUO. Most collecting ducts recovered following release. Our study indicates that obstruction leads to significant tubular damage, expansion of interstitial pericytes, fibrosis, tubular loss and formation of atubular glomeruli. The striking recovery observed after release of ureteral obstruction suggests a reversal of cell fate changes and tubular regeneration. Elucidation of the cellular and molecular mechanisms mediating these events may be of use in the design of strategies for the prevention and/or treatment of kidney diseases and secondary hypertension.
Abstract 062: Regulation of Nephron Afferent Arteriole Resistance in Obesity: Role of Connecting Tubule Glomerular Feedback
Authors: Monu Sumit R , Maheshwari Mani , Wang Hong , Peterson Ed , Carretero Oscar
Published: June 2021

In obesity, renal damage is caused by increase in renal blood flow (RBF), glomerular capillary pressure (P
GC
), and single nephron glomerular filtration rate but the mechanism behind this alteration in renal hemodynamics is unclear. P
GC
is controlled mainly by the afferent arteriole (Af-Art) resistance. Af-Art resistance is regulated by mechanism similar to that in other arterioles and in addition, it is regulated by two intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to an increase in sodium chloride (NaCl) in the macula densa,
via
sodium–potassium-2-chloride cotransporter-2 (NKCC2) and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation and is mediated by connecting tubule
via
epithelial sodium channel (ENaC). CTGF is blocked by the ENaC inhibitor benzamil. Attenuation of TGF reduces Af-Art resistance and allows systemic pressure to get transmitted to the glomerulus that causes glomerular barotrauma/damage. In the current study, we tested the hypothesis that TGF is attenuated in obesity and that CTGF contributes to this effect.

We used Zucker obese rats (ZOR) while Zucker lean rats (ZLR) served as controls. We performed
in-vivo
renal micropuncture of individual rat nephrons while measuring stop-flow pressure (P
SF
), an index of P
GC.
TGF response was measured as a decrease in P
SF
induced by changing the rate of late proximal perfusion from 0 to 40nl/min in stepwise manner.CTGF was calculated as the difference of P
SF
value between vehicle and benzamil treatment, at each perfusion rate.

Maximal TGF response was significantly less in ZOR (6.16 ± 0.52 mmHg) when compared to the ZLR (8.35 ± 1.00mmHg),
p<0.05
, indicating TGF resetting in the ZOR. CTGF was significantly higher in ZOR (6.33±1.95 mmHg) when compared to ZLR (1.38±0.89 mmHg),
p<0.05
. When CTGF was inhibited with the ENaC blocker Benzamil (1μM), maximum P
SF
decrease was 12.30±1.72 mmHg in ZOR and 10.60 ± 1.73 mmHg in ZLR, indicating that blockade of CTGF restored TGF response in ZOR.

These observations led us to conclude that TGF is reset in ZOR and that enhanced CTGF contributes to this effect. Increase in CTGF may explain higher renal blood flow, increased P
GC
and higher glomerular damage in obesity.
Abstract 063: Role of Ribosomal S6 Kinase 2 in Vascular Myogenic Tone and Blood Pressure Regulation
Authors: Artamonov Mykhaylo , Good Miranda , Momotani Ko , Isakson Brant , Carey Robert , Le Thu H , Somlyo Avril V
Published: June 2021

Smooth muscle (SM) contractile force is generated through phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase. However, emerging evidence also suggests that additional Ser/Thr kinases may contribute to the pathways that regulate SM contractility. Recently, we published that the 90 kDa ribosomal S6 kinase (RSK), extensively studied in cancers of epithelial origin, is a major new player in the regulation of SM contractility. RSKs are unusual kinases having two catalytic domains separated by a linker having multiple phosphorylation sites and are activated by a cascade of ERK phosphorylations and by PDK, which phosphorylates RSK at Ser227 resulting in transition to a fully active enzyme. The thromboxane agonist, U46619 leads to activation of ERK1/2 and PDK1 in SM. We find that 4
th
order mesenteric arteries are significantly more dilated at a pressure of 80 mm Hg in

Rsk2
-/-

mice compared to wild-type (WT) (2.0±0.8 vs 12.8±4.5 %,
p
<0.05, respectively). The contractile response to dose-dependent phenylephrine stimulation is significantly smaller in the absence of RSK2,
p
<0.0001 two-way ANOVA, while EC50 for phenylephrine contractions did not differ. Using isolated arcades of mesenteric vessels, phosphorylation of myosin RLC20 and myosin phosphatase regulatory subunit and RSK Ser227 (PDK site) and Thr 577 (ERK site) increased with in 30secs following an increase in intraluminal pressure to 80 mm Hg to induce myogenic tone development. Mean baseline systolic blood pressure (SBP, mmHg) was significantly lower in
Rsk2
-/-
animals (WT=107.4±1.6 and

RSK2
-/-

=95.8±2.2, n=23, p< 0.0002). In view of the lower SBP in the

Rsk2
-/-

animals, we examined the potential role for RSK signaling in the kidney, since RSK2 is known to phosphorylate NHE3. By immunohistochemistry, we show for the first time the expression of RSK2 in human kidney tubules. Lithium excretion, moles/kg/day, was increased in Rsk2-/- mice compared to WT (21.7±3.7 vs 48.6±7.9, respectively), indicative of defective renal Na
+
reabsorption. Taken together, RSK2 signaling in vascular SM and the kidney is a novel mechanism for the regulation of SM contractility and of BP. RSK2 may be a potential selective target in hypertension.
Abstract 064: Role of Sphingosine-1-phosphate 2 Receptor in Renal Microvascular Function of Renal Ischemia-reperfusion Rats
Authors: Guan Zhengrong , Inscho Edward W
Published: June 2021

Renal ischemia-reperfusion (IR) induced acute kidney injury (IR-AKI) is accompanied by increased renal vascular resistance and a dramatic decline in glomerular filtration rate. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in IR-AKI. Our recent studies established that exogenous S1P is a potent vasoconstrictor in rat afferent arterioles. We postulated that renal IR enhanced sensitivity of afferent arterioles to S1P-induced vasoconstriction via S1P2 receptor activation. The afferent arteriole response to S1P was assessed using the
in vitro
blood-perfused juxtamedullary nephron preparation. IR rats were induced by 60 min bilateral renal artery occlusion followed by 24 hours reperfusion. Baseline arteriolar diameter decreased significantly in IR (11.9±0.7 vs. 14.7±0.5 μm in sham, n=7, P<0.05). Exogenous S1P evoked concentration-dependent vasoconstriction in sham rats. Increasing S1P (10
-10
-10
-5
M) decreased diameter to 99±1, 95±2, 89±2, 80±3, 58±4, and 34±2% of baseline (n=7), respectively. Renal IR shifted the S1P vasoconstrictor profile to the left. Arteriolar diameter declined to 93±1, 87±1, 76±1, 64±5, 46±5 and 35±4% of baseline (n=7, P<0.05 vs sham at 10
-10
-10
-7
M S1P). S1P2 receptor blockade with JTE-013 markedly increased baseline diameter by 51±13% in IR (P<0.05, vs. 11±3% in shams, n=3-4), suggesting that endogenous S1P exerts a greater influence on arteriolar tonic in IR than in sham. JTE-013 also markedly attenuated the enhanced vasoconstriction to S1P in IR rats. Arteriolar diameter averaged 112±2, 110±1, 114±3, 102±6, 81±6 and 61±10% of control in response to increasing S1P (P<0.05 vs. JTE-013 untreated-IR) similar to JTE-013 treated sham. In contrast, arteriolar responses to Ang II, endothelin-1 or the S1P precursor, sphingosine were unaltered in IR rats. S1P content increased from 1.3±0.4 to 1.9±0.4 pmol/mg protein (~45%) in the cortical tissue of IR rats and 2.2±0.4 to 3.4±0.2 pmol/mg protein (~55%) in the medullary tissue but reduced the plasma S1P concentration from 2.5±0.3 to 1.3±0.1 μM (~50%, P<0.05, n=3). These data establish that renal IR increases sphingolipid metabolites in rat kidneys and enhances renal microvascular S1P signaling, probably via S1P2 receptor activation.
Abstract 066: Enhancer Repertoires That Define Renin Cell Identity
Authors: Martinez Maria F , Medrano Silvia , Oka Masafumi , Pentz Ellen S , Dickerman Allan W , Adli Mazhar , Sequeira-Lopez Maria Luisa S , Gomez R A
Published: June 2021

Control of the renin cell phenotype is crucial for the regulation of blood pressure and fluid- electrolyte homeostasis. Enhancers are
cis
-acting DNA sequences that harbor distinct chromatin features and regulate gene expression in an orientation-independent manner. Recently, clusters of enhancers or super-enhancers (SE) highly enriched with master transcription factors, possessing open chromatin configuration and in close proximity to cell-identity genes have been proposed. We tested the hypothesis that renin cells have unique repertoires of enhancers and super-enhancers, distinct from other cell types. Those regulatory clusters may in turn confer the identity of renin cells. To define the genome-wide enhancer landscape characteristic of renin cells, we studied As4.1 cells, kidney tumor cells that express renin constitutively, and native renin cells sorted from the kidneys of

Ren1cKO-YFP
+

mice. In these mice, the renin promoter drives YFP expression thus marking the renin cells. We used genome-wide ChIP-Seq for Med1 (subunit 1 of the Mediator complex), H3K27Ac (active enhancers) and Pol II (to visualize putative genomic areas undergoing transcription). The ROSE algorithm we used to ascertain super-enhancers. Chromatin accessibility genome-wide was assessed using ATAC-Seq. The results were compared to twenty-one other cell types that do not express renin. In As4.1 cells, we identified 14,871 enhancers based on H3K27Ac. Of those, 888 were classified as super-enhancers. The Med1 signal in As4.1 cells showed a SE localized 5kb upstream the
Ren1
gene, which was ranked at position 25 among other SEs. The H3K27Ac signal showed highest occupancy in the same region. ChIP-Seq for H3K27Ac in YFP
+
cells showed 211 SEs of 2,987 peaks. The SE for the renin gene possessed the highest signal and ranked number 1, indicating its importance in renin cells. One hundred and thirteen SEs were unique to renin cells, including the SE associated with the renin gene. ATAC-Seq signals overlapped with the renin SE and the classical enhancer indicating that the chromatin was accessible for transcription. In summary, renin-expressing cells possess distinct repertoires of unique enhancers and super-enhancers that acting in concert are likely to determine the renin phenotype.
Abstract 072: Brain Epithelial Sodium Channels on Organum Vasculosum of the Lamina Terminalis Neurons Mediate Sympathoexcitatory Pressor Responses and Neuronal Excitation by Hypertonic NaCl
Authors: Kinsman Brian J , Browning Kirsteen N , Stocker Sean D
Published: June 2021

High dietary salt intake raises cerebrospinal fluid (CSF) [Na
+
] in salt sensitive subjects to elevate sympathetic nerve activity (SNA), and arterial blood pressure (ABP). This occurs through excitation of NaCl-sensitive sites in the brain, including the organum vasculosum of the lamina terminalis (OVLT). Intriguingly, intracerebroventricular (ICV) pretreatment with benzamil (a non-voltage gated Na
+
channel blocker) also attenuates those systemic responses to central NaCl. Thus, I hypothesized that benzamil acts on NaCl-sensitive OVLT neurons to attenuate neuronal excitation and elevated SNA and ABP in response to hypertonic NaCl. To evaluate this hypothesis, lumbar SNA and ABP were measured in anesthetized adult rats in response to ICV infusion of 0.15, 0.5, and 1.0 M NaCl with and without prior OVLT microinjection of benzamil (5nmol per 20nL). ICV infusion of NaCl produced concentration-dependent increases in lumbar SNA (0.15M: 101±3%, 0.5M: 118±3%, 1.0M: 130±9% n=4 per group, P<0.05) and mean ABP (0.15M: 1±1mmHg; 0.5M: 5±1mmHg, 1.0M: 12±2mmHg). OVLT microinjection of benzamil significantly attenuated the increase in lumbar SNA (0.15M: 100±2%, 0.5M: 108±2%, 1.0M: 115±3% n=4 per group, P<0.05) and mean ABP (0.15M: 1±0mmHg; 0.5M: 2±1mmHg, 1.0M: 6±2mmHg). In a parallel set of experiments, in vitro whole-cell recordings of OVLT neurons in slices were performed to assess whether blockade of epithelial sodium channels (ENaC) attenuated NaCl-induced excitation. Bath application of +7.5mM NaCl increase action potential (AP) discharge of OVLT neurons (n=11) from 0.46±0.16 Hz to 1.09±0.26 Hz (P<0.05). Subsequent addition of an ENaC selective concentration of benzamil (0.5μM) reversed AP discharge (0.75±0.24 Hz, P<0.05). Isotonic 0.5μM benzamil did not significantly change NaCl-sensitive OVLT neuron (n=13) AP discharge from baseline (0.54±0.14 Hz to 0.60±0.16 Hz, P>0.05). Collectively, this data indicates that elevations in CSF NaCl concentrations excite OVLT neurons via ENaC to elevate SNA and ABP.
Abstract 073: Hindbrain Angiotensin Type-2 Receptors and Hypertension
Authors: de Kloet Annette D , Wang Lei , Ludin Jacob A , Hiller Helmut , Smith Justin A , Scheuer Deborah A , Steckelings Urlike M , Krause Eric G , Sumners Colin
Published: June 2021

The role of the angiotensin type-2 receptor (AT2R) in the neural control of cardiovascular homeostasis and hypertension is not well-characterized. Using a BAC transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse, dense localization of AT2R to GABA neurons was found within the nucleus of the solitary tract (NTS) of the hindbrain, an area important for regulating baroreflex function and blood pressure. This localization was confirmed by RNAscope fluorescence
in situ
hybridization. Considering that GABA is pressor in the intermediate NTS (intNTS), and that its effects are enhanced in models of neurogenic hypertension such as SHR and deoxycorticosterone acetate (DOCA)-salt rats, we tested the
hypothesis
that AT2R on GABA neurons in the NTS constitute a counter-regulatory, blood pressure lowering mechanism. In support of this idea, we have demonstrated that: (i) Optogenetic stimulation of GABA neurons in the intNTS of normal mice elicited a significant increase in blood pressure; (ii) mRNA levels for both the GABA synthetic enzyme Gad1 and for the AT2R are significantly increased in the intNTS of DOCA-salt hypertensive mice; (iii) Intracerebroventricular (ICV) infusion of the AT2R agonist Compound 21 (C21; 7.5 ng/h) into normotensive or DOCA-salt hypertensive mice elicited a significant reduction of systolic blood pressure, an effect that was much larger in hypertensive (126.2 ± 5.0 v. 139.8 ± 3.2 mmHg; n = 14; p = 0.02) than in normotensive (119.8 ± 2.6 v. 126.5 ± 2.5 mmHg; n = 16; p = 0.04) mice and was accompanied by decreased levels of the GABA synthetic enzymes Gad1 and Gad2 in the intNTS; (iv) Finally, the crucial involvement of GABA neurons in the blood pressure lowering effect of AT2R was proven by the lack of any effect of ICV C21 in DOCA-salt hypertensive mice containing a selective knockout of AT2R from GABA neurons. These novel data provide strong evidence for an anti-hypertensive action of AT2R within the intNTS, an effect that is exerted through decreases in GABA transmission.
Abstract 074: Epigenetic Regulation of Brain (Pro)renin Receptor by Benzamil-sensitive Sodium Channel in DOCA-salt Hypertension
Authors: Jensen Dane D , Feng Yumei
Published: June 2021

Increased expression of the brain (pro)renin receptor (PRR) contributes to the development of salt-sensitive hypertension (SSH). Yet, the mechanisms that drive this increase in PRR expression are not known. We previously reported an increase in histone 3 lysine 4 trimethylation (H3K4me3), an epigenetic marker for gene activation, on the PRR promoter in SSH mice. We hypothesized that the increase in brain PRR expression is driven by epigenetic modifications linked to high-salt intake. To test our hypothesis, C57Bl/6J mice were treated with Sham (sham pellet + tap water for drinking) or DOCA-salt (50mg DOCA pellet + 0.9% saline) with chronic intracerebroventricular (ICV) infusion of the epithelial sodium channel (ENaC) blocker Benzamil (2.64μg/day, 0.5μl/h) or aCSF for 3 days. Hypothalamic tissues were used to examine the chromatin modifiers activity (ng/h/mg of protein) responsible for the methylation or demethylation of H3K4 including the histone methyltransferase (HMT) and histone demethylase (HDM). Water intake and PRR mRNA level were monitored. In mice receiving ICV aCSF, DOCA-Salt treatment increased the activity of H3K4 HMT (18.5 ± 1.2 vs. 6.6 ± 0.3, P<0.001) and HDM (0.55 ± 0.03 vs. 0.37 ± 0.05, P<0.05) compared to Sham respectively. HMT activity was blunted by ICV infusion of Benzamil (14.2 ± 0.5, P<0.01) compared to ICV infusion of aCSF following 3 days of DOCA-salt treatment. Interestingly, HDM activity was further elevated with the treatment of ICV Benzamil compared to ICV infusion of aCSF (0.84 ± 0.03, P<0.01). ICV infusion of Benzamil also reduced the PRR mRNA levels in the hypothalamus of mice treated with DOCA-salt from (1.43 ± 0.09, P<0.05) to (1.07 ± 0.02 P>0.05) relative to Sham (1.00). In addition, the saline intake amount (ml/day) was lower (P<0.05) in mice receiving ICV infusion of Benzamil (17 ± 3) compared to ICV aCSF (32 ± 9) following DOCA-salt treatment. In summary, ICV infusion of Benzamil attenuates the elevation in HMT activity, water and sodium intake, and the PRR mRNA levels, while increasing HDM activity in DOCA-salt treated mice. We conclude the Benzamil-sensitive sodium channel, possibly ENaC, may be a key channel regulating the epigenetic modifications governing the increased expression of brain PRR in SSH.
Abstract 075: Impaired Hypothalamic PVN G-alpha i
2
Signal Transduction Attenuates Endogenous Sympathoinhibitory PVN GABAergic Tone to Evoke the Development of Salt-sensitive Hypertension
Authors: Wainford Richard D , Shim Joon
Published: June 2021

Aim:
We hypothesize hypothalamic PVN Gαi
2
proteins, which are up regulated during high salt intake, facilitate sympathoinhibition and blood pressure regulation via mediating GABA
B
receptor signal transduction to maintain salt-resistance in the Sprague Dawley (SD) rat.

Methods:
Groups of male salt-resistant SD rats received a bilateral PVN infusion of a scrambled (SCR) or Gαi
2
oligodeoxynucleotide (ODN-300ng/side/day) and a normal 0.6% (NS) or high 4% NaCl (HS) diet for 7-days. On day-7 24h Na
+
balance was assessed - in sub-groups MAP, plasma norepinephrine (NE) content, PVN GAD67 [marker of GABAergic expression] and vGLUT2 [marker of glutamatergic expression] immunoreactivity, and the cardiovascular responses to bilateral administration of the GABA
B
receptor antagonist (CGP52432; 3nmol/side) and the ionotropic glutamate receptor antagonist (kynurenate; 1.4 μg/side) was assessed (N=4/gp/study).

Results:
In control SCR ODN PVN infused SD rats HS intake suppressed plasma NE (plasma NE [nmol/L] SCR NS 76±7 vs HS 43±6, P<0.05) and evoked increased GAD67, but not vGLUT2, immunoreactivity (Medial Parvocellluar (MP) GAD67 [relative fold change (normalized to DAPI) 8.4±1.1) in all PVN parvocellular sub-regions without impacting MAP or sodium balance. Following ODN-mediated PVN Gαi
2
down-regulation HS intake caused salt-sensitive hypertension (MAP [mmHg] Gαi
2
ODN NS 128±3, HS 148±4, P<0.05), failed to increase GAD67 expression and increased PVN vGLUT2 immunoreactivity in multiple PVN parvocellular sub-regions (MP vGLUT2 [relative fold change (normalized to DAPI) 3.2±0.8). In separate groups of rats bilateral PVN GABA
B
antagonism increased MAP in control SCR but not Gαi
2
ODN infused rats maintained on a HS intake (peak ΔMAP [mmHg] SCR ODN HS +17±3, Gαi
2
ODN HS +2±2). In contrast, bilateral PVN glutamate antagonism did not alter MAP in control SCR ODN infused rats but decreased MAP in hypertensive Gαi
2
ODN infused SD rats on a HS intake (peak ΔMAP [mmHg] SCR HS +3±3, Gαi
2
ODN HS -13±2).

Conclusion:
These data suggest impaired Gαi
2
signaling attenuates sympathoinhibitory PVN GPCR-mediated GABAergic tone (i.e., modulates GPCR coupled GABA
B
responses) and enhances sympathoexcitatory glutamatergic tone to evoke salt-sensitive hypertension in the SD rat.
Abstract 080: mTORC1 as a Novel Regulator of Vascular Endothelial Function in Obese Mice and Humans
Authors: Reho John J , Guo Deng-Fu , Olson Andrew , Wegman-Points Lauren , Samuel Isaac , Nau Peter , Smith Jessica , Pierce Gary L , Rahmouni Kamal
Published: June 2021

Obesity-induced hypertension is associated with vascular endothelial dysfunction. Recently, our laboratory has demonstrated a critical role of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in cardiovascular regulation. Here, we tested the hypothesis that dysregulation of mTORC1 signaling is involved in the endothelial dysfunction associated with obesity in mice and humans. We found that diet-induced obese (DIO) mice that display vascular endothelial dysfunction as compared to lean controls have increased mTORC1 signaling in aortic lysates indicated by the elevated (p<0.05) phosphorylated levels of mTOR and its downstream signaling targets S6-kinase and the ribosomal S6 protein measured by Western blot. Increased vascular mTORC1 signaling in DIO mice was associated with increased aortic NOX2 mRNA expression (2.0±0.2 vs. 1.0±0.3AU in lean controls; p<0.05). Isolated abdominal subcutaneous adipose arterioles from non-diabetic obese (BMI ≥30 kg/m
2
; n=4; age 51±6 yrs; BMI 54±3 kg/m
2
) humans exhibited a strong trend towards increased phosphorylated S6 protein compared to normal-weight (BMI <30kg/m
2
; n=3; age 44±15 yrs; BMI 26±1 kg/m
2
) individuals (5.0±1.9 vs 0.8±0.4AU; p=0.12), suggesting increased vascular mTORC1 signaling in human obesity. Next, we used an adenoviral construct of a constitutively active (CA) S6-kinase (Ad-CAS6K) to enhance mTORC1 signaling. In mouse endothelial cells, Ad-CAS6K increased mRNA expression of oxidative stress (NOX1and NOX2) and inflammatory markers (IL-1β) and decreased endothelial NOS expression (p<0.05). Transfection of aortic rings with the Ad-CAS6K resulted in impairment in acetylcholine-induced relaxation (Max. relaxation: 67± 5 vs. 81 ±3%; p<0.05) without altering the relaxation evoked by sodium nitroprusside (Max. relaxation: 90±1% vs. 90±2%) recapitulating the vascular phenotype in obese mice. Taken together, our data demonstrate a novel role of the mTORC1 signaling pathway in the regulation of vascular endothelial function. Our data also implicate dysregulation of the endothelial mTORC1 signaling pathway in the endothelial dysfunction associated with obesity.