bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 020: A Mitochondrial Renin-Angiotensin System: Internalization of Angiotensinogen
Authors: Wilson Bryan A , Pirro Nancy T , Gwathmey TanYa M , Rose James C , Chappell Mark C
Published: July 2021

There is compelling evidence for actions of an intracellular renin-angiotensin system (RAS) in various cell organelles including the endoplasmic reticulum, nucleus and the mitochondria (Mito). Indeed, angiotensin (Ang) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively in a previous study. Since elucidation of mitochondrial pathways for expression of RAS protein components as well as Ang II or Ang-(1-7) is equivocal at this time, we undertook a biochemical analysis of the Mito RAS from adult male sheep kidney. Cortical Mito were isolated by differential centrifugation and a discontinuous Percoll gradient. Purified Mito were co-enriched in the voltage-dependent anion channel, an outer Mito membrane marker as well as ATP synthase, an inner membrane marker. Angiotensinogen (Aogen; 55 kDa) was detected in Mito extracts by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the Ang I N-terminus. Two different renin antibodies identified a major 35 kDa protein band in the isolated Mito. Using the Ang I-directed Aogen antibody, active renin was confirmed by hydrolysis of Aogen that was abolished by aliskiren; however, trypsin exposure did not increase renin activity in the Mito. A pro-renin receptor (PRR) antibody failed to identify proteins in three Mito preparations, but revealed a prominent band in renal cortical membranes that corresponds to the size of PRR. Angiotensin peptides were quantified by three direct RIAs; the Mito content of Ang II and Ang-(1-7) were higher as compared to Ang I [23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; p<0.01, n=3]. Additionally, both neprilysin and thimet oligopeptidase activities that processed Ang I to Ang-(1-7) were evident. Finally, cortical Mito internalized radiolabeled Aogen at a rate of 33 ± 9 fmol/min/mg protein (n=3) at 37°C. The subsequent analysis of the labeled Mito by SDS-gel fractionation revealed a predominant radioactive band of 55 kDa for Aogen. Collectively, our data suggest that the internalization of Aogen and subsequent processing by active renin may yield des-[Ang I]-Aogen and the active peptides Ang II and Ang-(1-7) that may potentially contribute to mitochondrial function within the kidney.
Abstract 035: Differential Sodium-evoked Activation of Pvn Magnocellular vs. Parvocellular Neurons in Rats Lacking Central GαI<SUB.2 Proteins Contributes to Sustained Elevations in Blood Pressure
Authors: Carmichael Casey Y , Wainford Richard D
Published: July 2021

Aim:
To determine the role of brain Gαi
2
proteins in mediating sodium-evoked PVN neuronal activation and blood pressure regulation in conscious rats.

Methods:
24-h intracerebroventricular scrambled (SCR) or Gαi
2
oligodeoxynucleotide (ODN; 25μg/5μl)-pretreated conscious Sprague-Dawley rats were monitored for changes in MAP in response to HS (IV 3M NaCl; 0.14 ml/100g). Rats were sacrificed at control (C), 10, 40, or 100-min post-HS for PVN cFos IHC and analysis of plasma AVP and NE. Separate groups received a V
1a
receptor antagonist (IV; 10 μg/ml/kg) 5-min prior to HS.

Results:
No difference was observed in sodium-evoked peak change in MAP and MAP remained elevated at 100-min in Gαi
2
but not SCR ODN rats (MAP 100-min post-HS [mmHg] SCR 134±2
vs
Gαi
2
146±3, P<0.05). Significant increases in the number of Fos
+
PVN magnocellular neurons were observed post-HS in SCR and Gαi
2
ODN groups ([Fos
+
cells] SCR C 3±1
vs
100-min 31±5; Gαi
2
C 2±0
vs
100-min 26±4,
P
<0.05). A rapid increase in circulating AVP was observed at 10-min in both SCR (plasma AVP [pg/mL] C 12.2±1.6
vs
10-min 62.8±6.9,
P
<0.05) and Gαi
2
ODN (plasma AVP [pg/mL] C 12.1±1.5
vs
10-min 67.7±7.7
P
<0.05) groups and returned to control levels at 40- and 100-min. SCR ODN rats exhibited significant increases in the number of Fos
+
PVN parvocellular neurons ([Fos+ cells] C 15±1
vs
100-min 67±4,
P
<0.05) and a significant suppression in circulating NE post-HS (plasma NE [nmol/L] C 44.1±4.9
vs
10-min 17.4±3.9,
P
<0.05). Gαi
2
ODN rats exhibited significantly less Fos
+
parvocellular neurons compared to SCR ODN rats (100-min [Fos+ cells] SCR 67±4
vs
Gαi
2
30±2,
P
<0.05) and failed to suppress circulating NE (
P
>0.05). V
1a
receptor blockade prevented a HS-evoked increase in MAP in SCR ODN rats while Gαi
2
ODN rats exhibited elevated MAP (MAP 100-min [mmHg] SCR 125±2
vs
Gαi
2
135±0,
P
<0.05).

Conclusion:
Brain Gαi
2
proteins are required to mediate sodium-evoked parvocellular sympathetic, but not magnocellular vasopressinergic, responses to maintain physiological blood pressure regulation. A significant component of blood pressure control in this setting is regulated by the sympathetic nervous system, as supported by the attenuated activation of PVN parvocellular neurons and a failure to suppress circulating levels of NE in Gαi
2
ODN rats.
Abstract 053: Uncoupling the Metabolic and Cardiovascular Actions of Leptin through mTORC1 Signaling
Authors: Bell Balyssa B , Morgan Donald A , Rouabhi Mohamed , Rahmouni Kamal
Published: July 2021

The adipocyte-derived hormone leptin has a well-established role in the regulation of energy homeostasis, acting in the brain to decrease food intake and promote energy expenditure. Additionally, leptin increases regional sympathetic nerve activity (SNA) and arterial pressure. Multiple intracellular signaling cascades are activated by leptin via its long form receptor (LRb), but the specific roles of these pathways in mediating leptin’s various effects have not been fully understood. Recent evidences suggest that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in mediating leptin action. To determine the contribution of mTORC1 to the metabolic and cardiovascular effects of leptin, we generated conditional knockout mice that lack the critical mTORC1 subunit, Raptor, specifically in LRb-expressing cells (LRb
Cre
/Raptor
fl/fl
). Interestingly, body weight was comparable between LRb
Cre
/Raptor
fl/fl
mice and controls (29.6±0.8 g vs 31.0±0.8g at 14 weeks of age). Moreover, leptin treatment (1μg/g bw, intraperitoneally, twice daily for 4 days) led to a similar decrease in food intake (-1.6±0.8 g in LRb
Cre
/Raptor
fl/fl
mice vs -1.1±1.7 g in controls) and body weight (-5.9±0.8% vs -5.7±0.7%) in both groups. Next, we measured arterial pressure using radiotelemetry at baseline and in response to 2 μg intracerebroventricular (ICV) leptin. Baseline mean arterial pressure (MAP) was comparable between LRb
Cre
/Raptor
fl/fl
mice (108±9 mmHg) and controls (103±7 mmHg). However, ICV leptin significantly increased MAP in control mice (30±14 mmHg), but not in LRb
Cre
/Raptor
fl/fl
mice (1±9 mmHg, P<0.05 vs controls). The same pattern was observed for systolic and diastolic arterial pressure. Consistent with leptin’s action on MAP, we observed a significant increase in renal SNA in response to ICV leptin in control littermates (106±20%) that was absent in LRb
Cre
/Raptor
fl/fl
mice (-28±11%, P<0.05 vs controls) as determined by multifiber sympathetic nerve recordings. Our data suggest a critical role for mTORC1 signaling in mediating the cardiovascular sympathetic but not the metabolic actions of leptin, a dissociation that may have important implications for obesity-associated hypertension.
Abstract 073: Expression of Axl in Innate Immune Cells Contributes to Kidney Dysfunction and Onset of Hypertension
Authors: Korshunov Vyacheslav A , Ko Kyung A , Mickelsen Deanne , Wood Ronald W , Batchu Sri N
Published: July 2021

Introduction:
We previously reported that expression of the receptor tyrosine kinase Axl in hematopoietic cells is critical for kidney dysfunction in early hypertension. Here we investigated the role of Axl expression in innate immune cells in deoxycorticosterone acetate (DOCA)-salt induced hypertension.

Methods and Results:
RAG1-/- mice lack adaptive immune cells and displayed the same (~25 mmHg) increase in systolic blood pressure (BP) as C57BL/6J mice after 1 week of DOCA-salt. While in metabolic cages RAG1-/- drank more (14.3±0.9 mL) than C57BL/6J mice (10.6±2.5 mL) per day after 1 week of DOCA-salt. Ultrasound imaging confirmed that RAG1-/- had ~20 % larger kidneys vs. C57BL/6J mice after DOCA-salt. RAG1-/- kidneys accumulated 2 times more fluid (2.8±0.1 %) compared to C57BL/6J mice (1.4±0.5 %) after DOCA-salt. Flow cytometry on kidneys from RAG1-/- confirmed absence of T and B lymphocytes, while DOCA-salt increased presence of macrophages (1.1±0.3 x10
9
) compared to C57BL/6J mice (0.6±0.1 x10
9
). We successfully generated Axl/RAG1 double knockout mice and subjected the littermates to 1 week of DOCA-salt. Increases in systolic BP were the same in Axl/RAG1+/+ and Axl/RAG1-/- littermate mice. No differences were found in kidney volumes between the Axl/RAG1 genotypes as well. However, 24 hrs excretion volumes increased in Axl/RAG1-/- (50±6 %) compared to Axl/RAG1+/+ (31±6 %) littermates. Finally, renal artery blood flow velocity (611±52 mm/s) and resistive index (0.62±0.03) were reduced in Axl/RAG1+/+ but not in Axl/RAG1-/- mice (665±45 mm/s and 0.68±0.01, respectively) when compared to their controls.

Conclusions:
Our findings suggest that mice lacking lymphocytes compensate by increasing kidney macrophages that contribute to initial increase in BP. Depletion of Axl in innate immune cells partially reverses kidney dysfunction by improving renal artery function in early hypertension.
Abstract 084: Significant Roles of Hyperglycemia and Obesity in the Disruption of Blood Pressure Circadian Rhythm in Leptin-receptor Mutant Db/db Mice
Authors: Hou Tianfei , Su Wen , Gong Ming , Guo Zhenheng
Published: July 2021

Blood pressure (BP) exhibits 24-hour rhythm. Loss of BP oscillation has been found in up to 75% diabetic patients and is associated with increased risks of target organ injuries. However, the mechanisms underlying the disruption of BP circadian rhythm in diabetes remain poorly understood. We and others have demonstrated that type 2 diabetic
db/db
mice in C57/KsJ background have hypertension and severely disrupted BP circadian rhythm. Since these
db/db
mice were severely hyperglycemic (>600 mg/dL) as well as obese, it is unclear which factor or both contribute to the disruption of BP oscillation. Moreover, it is unclear whether clock genes are involved in the diabetes associated disruption of BP circadian rhythm. To address these specific questions, we cross bred the leptin receptor mutated
db/db
mice in the C57BL/KsJ background with PERIOD2::LUCIFERASE knock in mice in C57BL/6J background. At 4-5 months old, the blood glucose in these
db/db
-Per2 mice was higher than controls (320.3 vs 153 mg/dL) but was significantly lower than the C57/KsJ -
db/db
mice (608.5 mg/dL). However, the body weight of these
db/db
-per2 mice was significantly higher than both the C57/KsJ-
db/db
(66.4 vs 44.8 g) as well as control mice (33.9 g). The metabolic flexibility, which is represented by respiratory exchange ratio and measured using TSE LabMaster Indirect Calorimetry System, was significantly compromised in the
db/db
-per2 mice when compared to controls. We then determined the BP in the
db/db
-per2 mice using radiotelemetry under 12: 12 light: dark cycle. The circadian parameters of BP, including period length, amplitude and acrophase were calculated using Chronos-fit software. The results demonstrated that
db/db
-per2 mice have normal BP value but disrupted BP circadian rhythm, with decreased power of 24h oscillation, diminished amplitude and shifted acrophase. However, the extent of the disruption was significantly less than that we have reported in the C57/KsJ-
db/db
mice. By using LumiCycle, we are currently investigating the clock gene functions in various tissues including SCN, aorta, liver, and etc isolated from
db/db
-Per2 mice. In summary, we demonstrated that both hyperglycemia and obesity significantly contribute to the disruption in BP circadian rhythm in
db/db
mice.
Abstract 085: Role of Suppressor of Cytokines Signaling 3 (Socs3) in Modulating Chronic Metabolic and Cardiovascular Effects of Leptin
Authors: do Carmo Jussara M , Freeman John N , da Silva Alexandre A , Wang Zhen , Hall John E
Published: July 2021

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of leptin signaling. Hypothalamic SOCS3 is upregulated in obese animals fed a high-fat diet and has been suggested to contribute to development of resistance to leptin’s anorexic effects. In this study we determined whether deletion of SOCS3 in the entire central nervous system (CNS) amplifies the chronic anorexic and blood pressure (BP) effects of physiological increases in plasma leptin in mice fed a normal diet. SOCS3
flox/flox
-Nestin-cre mice were generated by breeding SOCS3
flox/flox
with Nestin-cre mice. BP and heart rate (HR) were recorded by telemetry, and oxygen consumption (VO
2
) was monitored by indirect calorimetry in 22-week-old SOCS3
flox/flox
-Nestin-cre (n=4) and control mice (SOCS3
flox/flox
, n=4). Compared to controls SOCS3
flox/flox
-Nestin-cre mice were lighter (30±1 vs 33±1 g) and normoglycemic (124±7 vs 146±10 mg/dl), consumed less food (3.0±0.4 vs 3.6±0.2 g/day) and had similar VO
2
(77±6 vs 73±3 ml/kg/min). SOCS3
flox/flox
-Nestin-cre mice had similar MAP (103±3 vs 107±3 mmHg) but higher HR (666±15 vs 602±17 bpm) compared to control mice. Chronic leptin infusion greatly reduced food in SOCS3
flox/flox
-Nestin-cre (46±3 vs 35±4%) and increased MAP (15±3 vs 7±2 mmHg) and VO2 (18±3 vs 14±2%) compared to control mice. No significant changes were observed in HR in either group. Leptin infusion significantly reduced blood glucose levels in both groups (124±7 to 97±7 vs 146±10 to 105±7 mg/dl). These results indicate that SOCS3 deletion in the entire CNS reduces body weight and food intake, and amplifies leptin’s effect on appetite and blood pressure and also suggest the SOCS3 signaling attenuates the chronic actions of leptin on blood pressure as well as appetite regulation even in non-obese mice fed a normal diet. (NHLBI-PO1HL51971, NIGMS P20GM104357 and AHA SDG5680016)
Abstract 089: CD36 Genetic Variant Impacts Nitric Oxide Regulation of Endothelial Function: Response to Chronic Treatment with Phosphodiesterase 5 Inhibition
Authors: Shibao Cyndya A , Celedonio Jorge E , Gregory-Love Latisha , Ramirez Claudia E , Arnold Amy C , Okamoto Luis E , Choi Leena , Biaggioni Italo , Abumrad Naji , Abumrad Nada N
Published: July 2021

CD36, a scavenger receptor expressed on endothelial cells, interacts with thrombospodin-1, a matrix protein that modulates nitric oxide-soluble guanylate cyclase (NO-sGC) signaling. CD36 genetic variants associate with endothelial dysfunction, atherosclerosis, hypertension and insulin resistance. A coding variant of CD36 (rs3211938, G/T genotype) that causes partial CD36 deficiency (50% reduction) is common (~18%) in African Americans (AA); however, it is unknown, if this genotype influences NO-dependent endothelial function. This study examined whether potentiating NO-sGC pathways with the phosphodiesterase 5 inhibitor, sildenafil citrate, improves endothelial function and insulin sensitivity in AA women with or without the G/T genotype. Forty-six AA women with metabolic syndrome (MetS) participated in a 4-week, parallel-arm, double-blind, and placebo-controlled study. Carefully matched subjects were randomly assigned to sildenafil citrate 20 mg TID versus placebo; sildenafil (n= 23, 42±10 years old, BMI 39±5 kg/m2, fasting insulin 15±8 uU/ml) and placebo (n=23, age 43±10, BMI 39±6 kg/m2, fasting insulin 14±10 uU/ml). Primary endpoints were insulin sensitivity and endothelial function measured by intravenous glucose tolerance test and flow mediated dilation, respectively. Treatment compliance was documented with plasma sildenafil levels (mean 57±50 ng/ml). There was no difference in insulin sensitivity (p=0.676) or flow-mediated dilation (p=0.649) between intervention groups. However, subgroup analyses showed a significant interaction between sildenafil citrate treatment and G/T genotype (p=0.018). Sildenafil citrate improved endothelial function in G/T carriers (the mean difference: 2.9, the 95% CI: -0.90 to 6.8, p = 0.126) and decreased endothelial function in T/T carriers (the mean difference: -2.6, the 95% CI: -5.1 to -0.1, p = 0.040). We conclude that the rs3211938 common CD36 genetic variant influences NO-dependent endothelial function in response to chronic treatment with phosphodiesterase 5 inhibition. Further studies are needed to determine if rs3211938 and other common CD36 genotypes influence endothelial function and the inter-individual variability in response to the drug.
Abstract 134: Activation of the Mineralocorticoid Receptor by O-glycosylation Through Hyperglycemia
Authors: Kuppusamy Maniselvan , Gomez-Sanchez Elise , Gomez-Sanchez Celso
Published: July 2021

Introduction:
Hypertension and diabetes are independent risk factors for cardiovascular disease, however why they frequently occur together is not clear. Inappropriate mineralocorticoid receptor (MR) activation is associated with diabetes and the metabolic syndrome, as well as hypertension and abnormal cardiovascular remodeling. MR antagonists are effective in reducing hypertension and delaying the onset of renal and cardiovascular complications in diabetes despite circulating aldosterone levels that are within normal limits. Glucose concentrations increase O-glycosylation of many proteins, thus alter their function. Hence, we hypothesized that increased O-GlcNac modification of the MR by high glucose enhances MR activation.

Methods:
MR transcriptional activity was studied in a mouse cortical collecting duct (M1) cell line stably transfected with a cDNA construct including the MR and one with a hormone-response element driving a Gaussia luciferase reporter gene. The cells were incubated for 48 h with low (5mM) or high (25mM) glucose media with and without Thiamet-G (TMG), an O-GlcNAcase inhibitor to inhibit deglycosylation, and 6-diazo-5-oxonorleucine (DON), a glucosamine-fructose-6-phosphate amidotransferase inhibitor (GFAT) to reduce O-GlcNAc levels. Additionally, MR and GR antagonists were used to identify receptor specificity under low and high glucose conditions. O-GlcNac-modified MR was co-immunoprecipitated with an MR antibody and detected with an O-GlcNAc antibody.

Results:
1. Co-immunoprecipitation assays showed that high glucose and TMG increased O-GlcNac-MR by 3-fold. 2. Compared to low glucose, treatment with high glucose and with TMG increased the transcriptional activity of MR by 300%. 3. DON decreased MR-reporter activity by 75%. 4. High glucose alone had no significant basal effect but significantly increased MR activation by aldosterone. 5. MR reporter activity was increased similarly by aldosterone and corticosterone.

Conclusion:
High glucose increased glycosylation of the MR, augmenting its transcriptional activity. Enhancement of MR activation by hyperglycemia may explain how MRs play a significant role in the cardiorenal pathology in Diabetes.
Abstract MP11: Striatin Heterozygous Knockout Mice Have Increased Aldosterone Sensitivity
Authors: Garza Amanda E , Pojoga Luminita , Baudrand Rene , Moize Burhanuddin , Yao Tham , Adler Gail K , Williams Gordon H
Published: July 2021

Background:
We recently demonstrated that mice lacking one copy of the striatin gene (Strn+/-) have salt sensitivity of blood pressure (BP) as compared with WT mice. To determine whether Strn+/- mice have increased sensitivity to aldosterone (ALDO), we assessed the effect on blood pressure of an ALDO infusion in WT and Strn+/- mice fed a liberal sodium diet.

Methods:
In this study we used 12 week old WT and Strn+/- littermate male mice. For each genotype, mice were placed on HS diet and randomized to either: 1) placebo 2) ALDO (200 μg/Kg/day) or 3) ALDO plus 100 mg/kg/day eplerenone. BP was measured by tail cuff plethysmography at baseline and after treatment. After 21 days of treatment, animals were placed in metabolic cages for 24 hours. Finally, animals were sacrificed and organs excised. Primary endpoints were BP, renal immunohistochemistry, protein analysis by western blot and mRNA expression by RT-PCR.

Results:
BP increased significantly in Strn+/- mice treated with ALDO (ΔBP: 12 ± 4 mmHg, p=0.03) but not placebo (ΔBP 6± 6 mmHg); the BP effect of ALDO was blunted by eplerenone (Δ 6 ± 3 mmHg). In contrast, none of the treatments had a significant effect on BP in WT mice. Kidney weight was significantly increased after 3 weeks of ALDO treatment in both WT and Strn+/- mice and this increase in kidney weight was prevented by treatment with eplerenone, with no difference between genotypes. WT mice had an increase in glomerular volume (GV) in HS/ALDO treated that was blunted by eplerenone. Interestingly, Strn+/- mice had increased GV across all 3 treatment groups compared with WT mice. pAkt/Akt ratios were reduced in Strn+/- mice versus WT mice across all treatments. Classic genomic MR targets (ENaC and SGK1) and non-genomic targets (pAkt/Akt) were significantly modulated in kidney tissue of Strn+/- mice compared to WT mice with chronic ALDO.

Conclusion:
Strn+/- mice have an increased sensitivity to infused ALDO (increased BP response and increased rise in renal ENaC and SGK1 protein) as compared to WT mice. Since loss of striatin directly reduces nongenomic not genomic action of ALDO, this study demonstrates for the first time that modifying the nongenomic pathway may under chronic, in vivo conditions led to increased sensitivity to the genomic actions of ALDO.
Abstract P003: Hyperactive Brain Renin Angiotensin System Induces Polydipsia Through mTORC1 Pathway
Authors: Muta Kenjiro , Morgan Donald A , Grobe Justin L , Sigmund Curt D , Rahmouni Kamal
Published: July 2021

Mechanistic target of rapamycin complex 1 (mTORC1) is a molecular hub for signaling pathways mediating a wide range of cellular events involved in the regulation of various physiological and pathophysiological processes. We previously demonstrated that Angiotensin II (Ang II) activates mTORC1 and its downstream effector ribosomal protein S6 kinase in neurons
in vitro
. Here, we investigated the role of brain mTORC1 in hypertension and polydipsia induced by Ang II. In wild-type mice, acute stimulation of angiotensin type 1 receptor signaling by intracerebroventricular (ICV) injection of Ang II (1 μg, 30 min) activated mTORC1 signaling in the subfornical organ (SFO), a critical brain region in cardiovascular control and fluid balance, as indicated by the significant increase in the number of phosphorylated S6-positive cells (32±2 vs 13±3 in vehicle group). Similar upregulation of the mTORC1 pathway in the SFO was also found in the mice treated subcutaneously with Ang II (1000 ng/kg/min) using an osmotic minipump for 1 week (27±3 vs 11±2 in vehicle group). To verify functional roles of the Ang II activation of mTORC1 in the SFO, we utilized hypertensive and polydipsic transgenic mice (sRA) that have a hyperactive brain renin-angiotensin system, resulting in SFO-overproduction of Ang II. Interestingly, sRA mice exhibited substantially elevated phospho-S6 immunoreactivity only in the SFO (64±6 vs 36±8 in controls) but not in other cardiovascular regulatory regions including the paraventricular nucleus. ICV delivery of mTORC1 blocker, rapamycin (10 ng/day for 7 days) significantly (p<0.05) reduced daily water intake (-4.5±0.7 mL) compared to vehicle-treated sRA mice (-0.7±0.6 mL). In contrast, systolic blood pressure remained unchanged with rapamycin treatment (123±1 vs 125±4 mmHg in pre-treatment) and was consistently higher than the control group (110±4 mmHg). These results suggest that mTORC1 activity in the SFO is a critical determinant of the polydipsia evoked by Ang II.
Abstract P005: Mitochondrial Dysfunction in Heart of Coronary Artery Disease: Correlation with Telomerase Activity
Authors: Ait-Aissa Karima , Kim Joohwan , Morgan Garrett , Santos Janine H , Camara Amadou K , Gutterman David D , Betts Dean H , Donato Tony , Beyer Andreas M
Published: July 2021

Rational:
Heart disease is the leading cause of death worldwide and abnormalities in mitochondrial function are increasingly recognized in association with cardiomyopathy, heart failure, endothelial dysfunction and coronary artery disease (CAD). However the direct contribution and mechanism of the mitochondrial dysfunction on the development of CAD is not fully determined. We have recently shown a critical role of TERT, the catalytic subunit of telomerase, as a regulator of mitochondrial integrity in the microcirculation. We observed increased expression of the dominant negative splice variant of TERT (
β
-del) in the Left Ventricle from subjects with CAD. Therefore,
we hypothesize that TERT (

β

-del) decreases mitochondrial telomerase activity in the human heart resulting in mitochondrial damage that contributes to an environment that promotes the development of CAD
.

Methods:
Fresh and frozen tissue samples of discarded heart tissue from subjects with and without CAD were used. Protein, cell lysate or mitochondria were isolated using standard techniques. Mitochondrial DNA, levels of NAD+ and ATP as well as mitochondrial oxidative phosphorylation were evaluated.

Results:
PCR analysis revealed an increased frequency of mitochondrial common deletion, an established marker for mitochondrial abnormalities (0.9±0.2 in CAD; vs 1.5±0.2 in non-CAD; N=8; P<0.05). NAD+ and ATP levels were significantly decreased in CAD subjects compared to non-CAD (291±62 and 0.5±0.1 RLU/mg protein in CAD vs. 4203±336 and 84.1±24.8 pmol/mg protein in non-CAD respectively; N=15; P<0.005). Decrease respiration control index (RCI) in the presence of either complex I substrate K (+)-pyruvate/malate (PM) or complex II substrate K (+)-succinate (SUC) was observed in tissue form subjects with CAD (KPM-RCI: 2.9±1.3; SUC-RCI: 7.6± 1.9 in CAD vs KPM-RCI: 8.5±1.9; SUC-RCI: 19.1± 8.3 in non-CAD; N=3; P<0.05)

Conclusions:
Together these results point to significantly impaired mitochondrial function in subjects with CAD that are associated with decreased in mitochondrial telomerase activity.
Abstract P022: Association of Longitudinal Change in Kidney Function with Left Ventricular Structure and Function as Measured by Cardiac MRI: The Multi-Ethnic Study of Atherosclerosis (MESA)
Authors: Ohyama Yoshiaki , Ambale-Venkatesh Bharath , Yoneyama Kihei , Donekal Sirisha , Kishi Satoru , Volpe Gustavo J , Bensal Nisha , Kestenbaum Bryan , Nakamura Tetsutya , Kramer Holly , Shlipak Micheal , Colin Wu O , Bluemke David A , Lima Joao A
Published: July 2021

Background:
This study examines the association of change in estimated glomerular filtration rate (eGFR) with change in left ventricular (LV) structure and function over 10 years of follow-up.

Method:
MESA participants with cardiac magnetic resonance imaging (CMR) measures and eGFR measured at both baseline (2000-2002, year-0) and the 10 year follow-up (2010-2012, year-10) exam were studied. Participants with cardiovascular disease (CVD) were excluded. LV mass and volume indexed to body surface area, LV ejection fraction (LVEF), and LV mass volume ratio (MVR) were determined by CMR. We stratified participants by the presence of eGFR < 60 ml/min/m2 at baseline and at year 10 into 4 groups; eGFR ≥60 at both year-0 and year-10 (reference), ≥60 at year-0 and <60 at year-10, <60 at year-0 and ≥60 at year-10, and <60 at both year-0 and 10. Multiple linear regression models were used to evaluate the association between eGFR groups and change in LV parameters (year-10 - year-0) after adjusting for the demographics, baseline CVD risk factors, change in risk factors, and baseline LV parameters.

Results:
2,722 participants (age 59 ± 9 years, 53% women, 42% White, 13% Chinese, 25% African American, 20% Hispanic) were included. Compared to the reference group, the group with eGFR ≥60 at year-0 and <60 at year-10 and the group with eGFR <60 at both year-0 and 10 group showed increasing MVR during 10 year follow-up. No difference in longitudinal change in LV mass or LVEF by eGFR groups were noted (Table).

Conclusion:
In this multi-ethnic cohort of adults without clinical CVD, reduced eGFR at baseline or during follow-up was associated with the development of concentric remodeling represented by increased MVR.
Abstract P029: Vendor Specific Differences in the Sprague Dawley Rat Strain Alter Baseline Blood Pressure and Body Composition and Influence the Impact of Slow Fetal Growth on Later Cardiovascular Risk
Authors: Dasinger John Henry H , Intapad Suttira , Backstrom Miles A , Carter Anthony J , Alexander Barbara T
Published: July 2021

Low birth weight is associated with increased risk for cardiovascular (CV) disease including hypertension in later life. We previously reported that reduced uterine perfusion (RUP) in timed pregnant Sprague Dawley (SD) dams purchased from Harlan induced intrauterine growth restriction (IUGR) associated with hypertension and enhanced sensitivity to angiotensin II (Ang II) in male rats at 4 months of age. In addition, male IUGR exhibited a two-fold increase in testosterone relative to control. Upon castration, hypertension and sensitivity to Ang II were abolished suggesting that programmed CV risk is testosterone dependent in the male IUGR. The aim of this study was to determine if vendor differences in the SD strain impact CV risk following a developmental insult. Timed pregnant SD rats were purchased from Charles River and underwent either RUP or sham surgery at day 14 of gestation. Birth weight was significantly reduced in IUGR relative to control (P<0.05). At 4 months of age, body weight remained significantly decreased in male IUGR relative to control, blood pressure did not differ when measured in conscious, chronically instrumented animals, and male control and IUGR from Charles River also exhibited a similar blood pressure response to acute Ang II. However, baseline blood pressures were higher in male control from Charles River versus blood pressure previously noted for male control from Harlan. Thus, these results suggest that vendor specific differences in the SD rat strain influence baseline CV risk and effect the developmental programming of CV risk following slow fetal growth.
Abstract P043: Estradiol Treatment Duration Significantly Impacts Renal Health in Midlife Ovariectomized Female Long Evans Rats
Authors: Zimmerman Margaret A , Trimmer Emma H , Daniel Jill M , Lindsey Sarah H
Published: July 2021

Current clinical recommendations are that hormone therapy for the treatment of menopausal symptoms be limited to a few years. Our lab previously reported that both transient (40 days) and sustained (80 days) estradiol (E2) treatment initiated immediately after midlife ovariectomy (OVX) in Long Evans rats similarly attenuated OVX-induced increases in blood pressure (BP). This study indicated that the benefits of E2 could be maintained long after treatment cessation. Therefore, the goal of the current study was to determine whether E2 duration similarly protected against renal damage. We hypothesized that both transient and sustained E2 treatment exert positive effects on renal health in midlife OVX Long Evans female rats. Female retired breeders underwent OVX at 11 months of age and received an implant of E2 or vehicle (veh). After 40 days, implants were replaced comprising the following groups: veh>veh (n=9), E2>E2 (n=7), E2>veh (n=10). Animals were placed in metabolic cages at both the 40 and 80 day treatment time points to measure proteinuria, urinary peptides, and creatinine excretion. At the end of the 80 day treatment tissue weights were obtained. Interestingly, kidney hypertrophy was significantly increased in the E2>E2 when analyzed as raw wet weight (veh: 1.0 ± 0.05 g; E2>veh: 0.95 ± 0.02; E2>E2: 1.2 ± 0.04; P<0.0001), or normalized to body weight (veh: 2.5 ± 0.1 mg/gBW; E2>veh: 2.5 ± 0.03; E2>E2: 2.9 ± 0.11; P<0.005). Creatinine excretion was not different between groups (veh: 7.0 ± 0.6 mg/day; E2>veh: 6.4 ± 0.4; E2>E2: 7.6 ± 0.7; P=0.29). However, there was a significant increase in proteinuria measured by the Bradford assay in the E2>E2 group (veh: 2.0 ± 0.8 mg/mgCr; E2>veh: 0.9 ± 0.3; E2>E2: 6.8 ± 2.6; P<0.05). The E2>E2 group also had a significantly lower percentage of urinary peptides (veh: 96 ± 0.8%; E2>veh: 97 ± 0.7%; E2>E2: 79 ± 8.1%; P<0.05), indicating impaired renal function. These results indicate that in midlife OVX Long Evans rats, sustained E2 lowered BP but exerted detrimental effects on kidney function. In contrast, transient E2 treatment lowered BP without renal impairment. Overall, our results indicate that the period of exposure to estradiol may be important for renal health in aging postmenopausal women.
Abstract P081: Tempol Decreases Blood Pressure in Aged Female SHR When Treated With Acetazolamide
Authors: Dalmasso Carolina , Maranon Rodrigo O , Patil Chetan N , Harris Andrew , Zhang Huimin , Wilcox Christopher S , Reckelhoff Jane F
Published: July 2021

Oxidative stress contributes to the development and maintenance of hypertension in male rodents, but studies in females have rarely shown a reduction in blood pressure (BP) with antioxidants. Tempol, a superoxide dismutase mimetic, decreases BP in young male SHR, but fails to reduce BP in either young or old female SHR, despite the fact that females have similar or higher levels of oxidative stress markers. The reason for the sex difference in the response to tempol remains unclear. Acetazolamide inhibits carbonic anhydrase in the proximal tubule thus increasing sodium delivery to the distal nephron, and thereby should increase distal oxidative stress. Acetazolamide was used to test the hypothesis that with increased sodium delivery to the distal nephron, tempol would reduce the BP in aging female SHR. Female SHR, 20-22 mos old, were divided into three groups (n=4-6/grp): Control (C), Acetazolamide (A), and Acetazolamide+Tempol (A+T). After baseline mean arterial pressure (MAP; telemetry), rats received vehicle (C) or acetazolamide (A and A+T). On day 8, rats in C and A+T groups were given tempol (30 mg/kg) for 11 days. Baseline MAP was similar (C: 170±7; A: 182±4; A+T: 172±6 mm Hg, p=NS). Tempol had no effect on MAP in C+T, but reduced MAP in A+T group (C+T: 169±1; A: 171±1; A+T: 151±5 mm Hg; p<0.005 A+T vs A, C+T). Basal renal oxidative stress measured by lucigenin chemiluminescence was not different in the groups; NADPH-stimulated oxidative stress was decreased in A+T compared to A and C+T (C+T: 641.8±72.2; A: 499.3±18.3; A+T: 406.2±56.3 RLU/mg/min; p<0.05, A+T vs A, C+T). Plasma total antioxidant capacity was increased by tempol only in A+T rats (C+T: 59.07±9.67; A: 69.01±4.66; A+T: 118.24±18.38 nmol/μl; p<0.05, A+T vs A, C+T). Thus tempol is capable of modestly reducing MAP in aging female SHR when proximal sodium reabsorption is blocked. The data suggest that oxidative stress-mediated BP control is dependent on increased sodium delivery to the distal nephron. Because hypertension in male SHR is attenuated with tempol alone, but not in females, taken together, the data suggest sex differences in sodium handling and thus localization of oxidative stress production in the kidneys of SHR. Supported by NIH-R01HL66072, PO1HL51971 (JFR), 14POST18640015 (ROM).
Abstract P092: Proliferation of Endogenous T-regulatory Cells Improves the Pathophysiology Associated with Placental Ischemia of Pregnancy
Authors: Ibrahim Tarek , Przybyl Lukasz , Harmon Ashlyn C , Cornelius Denise C , Cunningham Mark W , Amaral Lorena M , Faulkner Jessica L , Dechend Ralf , LaMarca Babbette
Published: July 2021

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with pro-inflammatory cytokines and decreased regulatory immune mechanisms such as Tregs, IL-10 and IL-4. We believe this decrease in immune regulatory mechanisms leads to an uncontrolled proinflammatory response which contributes to most of the pathophysiology associated with PE. The Reduced Uterine Perfusion Pressure, RUPP, rat model of induced placental ischemia exhibits similar characteristics as women with PE including high blood pressure, elevated pro-inflammatory cytokines and cells and decreased Tregs, IL-4 and IL-10. Therefore, we hypothesized that stimulating Tregs by administration of a superagonist (SA) would increase the Treg profile in the RUPP rats which could reduce pro-inflammatory cytokines and blood pressure. The RUPP procedure was performed at gestation day 14 (GD14); SA was administered intraperitoneally at GD15, GD18 carotid catheters inserted, and GD19 MAP and pup weight, serum and tissues were collected. MAP in NP rats was 91 +/- 3, 119.6 +/- 2 in RUPPs which was improved to 111 +/- 1 mmHg in RUPP+SA. Circulating FoxP3+ Treg cells were 6 +/- 1.7% in NP, 0.77% +/- 0.75 in RUPP rats but increased to 11% +/- 3 in RUPP+SA; IL-6 was 33.65+/- 3.4 in NP, 117.2 +/- 37.8 in RUPP, and 43.66+/- 6.1 pg/mL in RUPP+SA. Placental Pre-pro Endothelin-1 (PPET-1) was increased 44.42+/- 0.269 fold in RUPP compared to NP 1+/- 0.255, but was decreased to 18.78+/- 0.48 in RUPP+SA. These data suggest an important role for up-regulating Treg cells to enhance the immune regulatory interactions and lower the hypertension without causing further reduction in fetal weight in response to placental ischemia during pregnancy.
Abstract P094: Vasopressin Infusion in Mice During Pregnancy Results in Immune Alterations Consistent with Human Preeclampsia
Authors: Scroggins Sabrina M , Santillan Donna A , Pearson Nicole A , Sangren Jeremy A , Min James Y , Devor Eric J , Pierce Gary L , Leslie Kimberly K , Grobe Justin L , Santillan Mark K
Published: July 2021

The immediate and long-term maternal and fetal health complications of preeclampsia (PreE) have been linked to an aberrant T helper immune response and pro-inflammatory cytokine profile. AVP secretion is both stimulated by and stimulates pro-inflammatory cytokine secretion and activation of lymphocytes. Our AVP infusion mouse model of PreE closely replicates human PreE. The objective of this study was to investigate if AVP infusion induces the maternal-fetal immune alterations in PreE.

Immune responses were evaluated in wild-type C57BL/6 female mice chronically infused with AVP (24 ng/hr or saline s.c.) throughout pregnancy. By flow cytometry, we observed an increase in the frequency of IL-17 (1.2% vs. 2.6%, p<0.05), TNFα (0.9% vs. 1.6%, p<0.0001), and IFNγ (1.8% vs. 3.4%, p<0.05) expressing T cells in AVP dams. An increase in the frequency of IL-12 producing cells (1.0% vs. 2.4%, p<0.05) and elevated expression of co-stimulatory molecules on dendritic cells (DCs) from AVP dams suggested enhanced DC function. The maternal plasma revealed a significant reduction in IL-17 (7.1e+6 vs. 3.4e+5 ng/g, p<0.05) and IL-6 (3.4e+5 vs. 0.0 ng/g, p<0.01) production in AVP dams by ELISA as seen in human PreE. Pro-inflammatory TNFα was also significantly decreased in the maternal kidney of AVP dams (2.3e+9 vs. 1.3e+9 ng/g, p<0.05). IL-4 production was significantly reduced in the maternal kidney of AVP dams (5.7e+7 vs. 3.4+7 ng/g, p<0.05) suggesting a blunted Th2 response as seen in human PreE. AVP infusion did not result in alterations of cytokine production in the maternal liver, or fetal liver and kidney. Interestingly, IL-17 production was increased in the amniotic fluid (3.0e+6 vs. 1.3e+7 ng/g, p<0.05) and lower in the placenta (2.5e+7 vs. 1.3e+7 ng/g, p<0.0001) of AVP dams. IL-4 production was decreased in both the amniotic fluid (4.5e+5 vs. 2.0e+5 ng/g, p<0.05) and placenta (3.1e+6 vs. 2.0e+6 ng/g, p<0.05) of AVP dams again suggesting a lower Th2 response.

These data support our hypothesis that AVP induces maternal-fetal immune alterations consistent with human PreE. Ongoing experiments are aimed at identifying the lymphocytes and cytokines involved as well as local vs. systemic immune dysfunction induced by AVP in pregnancy.
Abstract P097: Molecular Mechanisms of VEGFR Inhibition-induced Endothelial Cell Damage
Authors: Rios Francisco J , Montezano Augusto C , Van Der Mey Lucas , Small Heather Y , Savoia Carmine , Touyz Rhian M
Published: July 2021

VEGF/VEGFR inhibitors, used as anti-angiogenic drugs to treat cancer, induce severe hypertension. Molecular mechanisms whereby VEGF inhibitors cause hypertension are unclear, but nitric oxide (NO) and oxidative stress may be involved. We questioned whether reactive oxygen species (ROS) and Ang II, important regulators of vascular function in hypertension, also play a role in VEGF inhibitor-induced vascular dysfunction. Human microvascular endothelial cells (HMECs) were stimulated with vatalanib (VAT-VEGFR inhibitor) and gefitinib (GEF-EGFR inhibitor) in the absence/presence of Ang II. Activation of eNOS and MAPKs were assessed by immunoblotting. Antioxidant enzyme mRNA was analysed by qPCR. Microparticle levels were measured by flow cytometry. Endothelial microparticles, biomarkers of endothelial damage, tend to increase in subjects treated with VEGFR inhibitors. Phosphorylation of eNOS activation site (Ser1177) (28.3% ± 7.1) was decreased by VAT, while no changes were observed after exposure of HMECs to GEF (p<0.05). VAT decreased mRNA expression of Nox4 (0.5 ± 0.2) and H2O2-regulating antioxidants enzymes such as catalase (0.4 ± 0.1) and glutathione peroxidase (0.4 ± 0.1), while increased mRNA levels of Nox5 (3.35±1.1) (p<0.05 vs. veh). Ang II stimulation increased eNOS (171.2% ± 17.4) and ERK1/2 (177.5% ± 38.5) activation (p<0.05); all effects were blocked only by GEF. Inhibition of VEGFR also blocked Ang II effects on SOD1 (1.33 ± 0.1), HO-1 (1.6 ± 0.3) and NQO1 (1.6 ± 0.3) mRNA levels (p<0.05). In addition, Ang II increased Nox4 mRNA expression through VEGFR-dependent mechanisms. VEGFR1/2 and AT2R, but not AT1R, were expressed in HMEC. Ang II effects on eNOS phosphorylation were inhibited by PD123319 (AT2R antagonist) but not by losartan (AT1R antagonist). In conclusion, our data identify novel mechanisms whereby AngII, possibly through AT2R-dependent VEGFR transactivation, regulates eNOS activation, MAPK signalling and H2O2-related antioxidant enzymes. In addition to changes in NO availability, VEGFR inhibition may interfere with the redox status of endothelial cells, leading to vascular dysfunction and hypertension.
Abstract P144: Effects of Alamandine in Post-ischemic Function
Authors: Almeida Jonathas F , Santos Robson A
Published: July 2021

Alamandine, a biologically active peptide of the renin-angiotensin system (RAS), was recently described and characterized. Further it has been shown to present effects similar to those elicited by Ang-(1-7). It has been described that Ang-(1-7) decreases the incidence and duration of ischemia-reperfusion arrhythmias and improved the post-ischemic function in isolated perfused rat hearts. In this study we aimed to evaluate the effects of Alamandine in isolated rat hearts subjected to myocardial infarction (MI). Wistar rats weighing between 250-300g were euthanized and their hearts were placed on Langendorff apparatus to evaluate the cardiac parameters. Hearts were submitted to 30min of stabilization, 30min of partial ischemia by occlusion of the left descending coronary artery and 30min of reperfusion. Drugs (alamandine 22pM, d-pro7-ang-(1-7) 220pM) were added to the perfusion setting from the beginning of the experiment until the end. 2,3,5-trypheniltetrazolium chloride were used to evaluate the extension of infarcted area. In control hearts (CON), there was a decrease on the left ventricular systolic pressure (LVSP) on ischemic period (54,6 ± 6,9mmHg) compared to the baseline period (84,6 ± 11,6mmHg). Alamandine (ALA) attenuated that decrease in the ischemic period (66,9 ± 7,9mmHg) vs (82,3 ± 8,9mmHg). Further, ischemia led to a decrease in the left ventricular developed pressure (dLVP), dP/dt maximum and minimum when compared to baseline values. ALA, once more, kept the ischemic parameters of dLVP and dP/dt max and min (58,9 ± 8mmHg; 1629 ± 202,2mmHg/s; 1101 ± 130mmHg/s, respectively) similar to those of baseline period (68,9 ± 8,92; 1682 ± 248,8; 1179 ± 118,6 mmHg, respectively). Ischemia/reperfusion induced an arrhythmia severity index (ASI) in control hearts (4,9 ± 1,26) higher than in hearts treated with ALA (1,10 ± 0,58). ALA also reduced infarcted area (19,64 ± 2,61%) compared with CON (33,85 ± 4,55%). All those effects were blocked by D-PRO7-Ang-(1-7). In conclusion, our data shown that Alamandine exert cardioprotective effects in post-ischemic function in isolated rat hearts by preventing LVSP, dLVP , dP/dt max and min decrease. Furthermore it reduced the infarcted area and I/R arrhythmias, apparently involving MrgD receptor participation.
Abstract P150: Podocyte Derived Urinary Microparticles Are Elevated in Angiotensin II Induced Hypertension
Authors: Erdbruegger Uta , Rudy Christine , Bennett Deric , Cechova Sylvia , Chang Fang , Le Thu H
Published: July 2021

Background:
Early and non-invasive biomarkers of kidney damage are needed to identify hypertensive patients at risk for kidney damage. Urinary micropaticles (UMPs) have gained significant attention as potential novel biomarkers for kidney damage, and have already been identified in pre-albuminuric diabetic glomerular injury. These vesicles are less than 1 micron in size and carry markers of the parent cell. We hypothesized that podocyte derived UMPs are elevated in angiotensin II-induced hypertension (HTN)

Methods:
Primary podocytes were isolated and grown in culture. Wild-type mice were treated with AII (400ng/kg/min) via mini-osmotic pumps. Untreated WT mice served as controls. 24 hour urines were collected after 5 days of AII treatment. Enumeration and phenotyping of MPs was done of podocyte culture supernatant and urine. Podocalyxin (Pcal), podoplanin (Ppla) and annexin 5 (AV) were used as surface markers.

Result:
Pcal and Ppla positive MPs as well as AV positive and negative MPs were detectable in supernatant from primary podocyte cultures. Compared to untreated controls (n=3), AII treated mice (n=2) had an increase in systolic blood pressure (SBP) by 33 mmHG (p=0.02). Despite similar urinary albumin/creatinine ratios between groups, there was a trend of higher levels of total numbers of Ppla and Pcal positive MPs in hypertensive mice compared to untreated (Figure 1). In addition, Annexin negative but Ppla and Pcal positive MPs were also numerically higher in hypertensive mice.

In conclusion, podocyte derived UMPs are detectable in AII HTN. These findings need to be confirmed in a larger group of animals. UMPs can be potential marker for kidney end-organ damage in HTN.