bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 551: Inhibition of Sphingosine-1-phosphate Receptor 2 Activation Protects against Renal Ischemia-Reperfusion Injury via Reduction of Reactive Oxygen Species
Authors: Guan Zhengrong , Singletary Sean T , Inscho Edward W
Published: March 2022

Sphingosine-1-phosphate (S1P) is a potent vasoconstrictor of the preglomerular microvasculature. Our recent studies indicate that S1P-mediated afferent arteriolar vasoconstriction is enhanced in ischemia-reperfusion (IR) induced renal injury and S1P2 receptor protein expression is increased in renal microvessels. Reactive oxygen species (ROS) play a critical role in the development of IR renal injury. We hypothesized that inhibition of S1P2 receptor activation protects against renal IR injury via ROS reduction. Renal IR injury was induced by bilateral renal artery occlusion for 60 min followed by 24 hrs reperfusion. In some rats, IR was induced during treatment with the selective S1P2 receptor antagonist, JTE-013 (0.1mg/kg BW, IP) prior to ischemia and repeated at the time of reperfusion. After reperfusion, plasma was collected and kidneys were harvested for histological analysis and ROS detection. Plasma creatinine (Cr) concentration was markedly increased in IR rats (4.2±0.1 mg/dl, n=6) compared to sham controls (1.1±0.1 mg/dl, n=5, p<0.05). JTE-013 treatment (n=4) significantly reduced plasma Cr in IR rats (2.0±0.3 mg/dl, p<0.05). Histological analysis revealed no significant cell damage in control kidney sections stained with hematoxylin and eosin whereas severe cell damage was detected in IR kidneys, including cell necrosis, disruption of the plasma membrane, nuclear pyknosis, cellular vacuolization, and the presence of luminal casts and sloughed cells in proximal tubules, particularly in the outer stripe of the outer medulla. Additionally, there was significant trapping of erythrocytes in the medulla. In contrast, JTE-013 treated IR rats showed less necrosis and cell damage in the proximal tubules. Renal ROS levels were assessed in frozen kidney sections using the fluorogenic probe, H
2
DCFDA. Fluorescence intensity was markedly increased in IR rats (18.7±2.4 in cortex and 19.0±2.5 in medulla) compared to control (10.9±0.7 and 9.6±0.7, p<0.05, respectively). JTE-013 treatment reduced ROS levels to values not different from control (13.5±1.1 and 15.4±1.5, p>0.05 vs. control, respectively). These data suggest that blockade of S1P2 receptors protects against renal IR injury by reducing tissue ROS accumulation.
Abstract 557: Chronic Compound-21 Infusion Induces Natriuresis in Wild-Type Mice but not in AT2 Receptor-Null Mice
Authors: Howell Nancy L , Kemp Brandon A , Padia Shetal H , Carey Robert M
Published: March 2022

Our previous studies have demonstrated that angiotensin (Ang) type-2 receptors (AT2R) mediate natriuresis by inhibiting Na+ reabsorption in the renal proximal tubule and that des1-aspartyl-Ang II (Ang III) is the preferred endogenous agonist for this response. Natriuretic responses to intrarenal Ang III infusion do not occur in the absence of concurrent systemic Ang type-1 receptor (AT1R) blockade or aminopeptidase N inhibition to prevent Ang III degradation. In the present study, we hypothesize that Compound 21 (C21), a highly selective non-peptide AT2R agonist, administered chronically in the absence of concurrent AT1R blockade can induce natriuresis in wild-type mice (WT; C57B6) and that this response does not occur in AT2R-null mice (AT2-null). WT and AT2-null (N=8/group) on normal diet (0.3% NaCl) were infused subcutaneously with C21 (300 ng/kg/min) or vehicle (V) 5% dextrose in water via osmotic mini-pump continuously for 7 days. Food/fluid intake and systolic blood pressure (SBP; via telemetry for 30 min at 8 am) were monitored daily and 24 h urine Na+ excretion (UNaV) was measured at baseline and infusion days 1, 4, and 7. Baseline UNaV in V-infused WT was 0.09 ± 0.01 μmol/min and did not change with V infusion. In C21-infused WT, UNaV increased from a baseline of 0.11 ± 0.01 to 0.13 ± 0.02 μmol/min on day 4 and to 0.17 ± 0.02 μmol/min on day 7 (F=15.0, P<0.0001 from V-infused WT and AT2-null). UNaV in both V- and C21-infused AT2-null decreased in parallel from a baseline of 0.11 ± 0.01 to 0.07 ± 0.01 μmol/min on day 4 and to 0.06 ± 0.01 μmol/min on day 7 (P<0.0001 from C21-infused WT). Dietary consumption was identical among the 4 groups of mice over the 7 day study. SBP was similar in the 4 groups during the control period. The results demonstrate that C21 induces natriuresis chronically in an AT2R-dependent manner in the absence of AT1R blockade and indicates that C21 may be a useful natriuretic/diuretic agent in disorders of fluid retention.
Abstract 56: Mast Cell Chymase: An Angiotensin II (Ang II)-Independent Therapeutic Target in the Post-Myocardial Infarction Heart
Authors: Tejada Thor , Zlatopolsky Max , Bhushan Shashi , Naqvi Nawazish , Abrink Magnus , Pejler Gunnar , Lefer David , Husain Ahsan
Published: March 2022

Mast cell chymase (CHY), a multifunctional protease with Ang II-forming activity, garnered interest due to the success of angiotensin converting enzyme inhibitors (ACEi) in the treatment of heart failure. The VALIANT trial evaluated the contribution of this alternate Ang II pathway by adding an AT
1
Ang II receptor blocker (ARB) to an ACEi post-myocardial infarction (MI), but the combination showed no additional benefit, suggesting CHY was unimportant post-MI. However, the role of CHY, independent of its Ang II-forming activity, remains unaddressed. To test this, we first re-evaluated the role of CHY-generated Ang II by blocking the effects of ACE+CHY-generated Ang II using an ACEi+ARB+AT
2
receptor blocker (PD122139) combination (AAA), and compared this to ACEi monotherapy in wild-type (WT) mice. We then addressed the Ang II-independent role of CHY by comparing these therapies in chymase (MMCP-4)-deficient (KO) vs WT mice. WT and KO mice (N=10-20) underwent sham or MI (90-min ischemia-reperfusion) surgery, and were treated daily with vehicle (Veh), ACEi, or AAA 24h post reperfusion. After 14 days, echocardiographic and hemodynamic analyses indicated no significant inter-genotype differences in sham animals. In post-MI WT mice, ACEi vs Veh resulted in improved ejection fraction (EF) (34±1.8% vs 28±1.7%; p<0.05) and reduced LV end-diastolic dimension (LVEDD) (4.2+0.09 mm vs 4.7±0.1 mm; p<0.05). However, AAA did not result in further improvements in either parameter as compared to ACEi, suggesting that CHY-generated Ang II is unimportant post-MI, confirming VALIANT. While the initial (24-h post-MI) infarct sizes were similar between genotypes (WT: 63±5% vs KO: 65±2% infarct as percent of area at risk; p=0.6), EF was superior in KO vs WT mice in all treatment groups (36±3% vs 28±2% (Veh); 47±3% vs 34±2% (ACEi); 49±3% vs 36±3% (AAA); p<0.05). Further, LV dilatation was also more pronounced in WT mice (LVEDD: 4.7±0.4 mm, WT-Veh vs 4.3±0.5 mm, KO-Veh; p<0.05). ACEi decreased mean arterial pressure post-MI (p<0.01), but not differentially in WT vs KO (Veh: 70±5 mmHg vs 66±3 mmHg; p=0.47; ACEi: 48±3.7 mmHg vs 38±4.8 mmHg; N=6−10; p=0.1). Thus, we conclude that CHY, independent of Ang II, is a potential therapeutic target for the treatment of the post-MI heart.
Abstract 568: Regulation of GRK4 on Arterial AT
1
Receptor in Hypertension
Authors: Chen Ken , Liu Li , Chen Caiyu , Zeng Chunyu
Published: March 2022

G protein-coupled receptor kinase 4 (GRK4) gene variants or increased GRK4 expression, via impairment of renal dopamine receptor and enhancement of renin angiotensin system function, impairs renal sodium excretion, resulting in sodium retention and an increase in blood pressure. Increased aortic stiffness, a risk factor in cardiovascular disease, may be related to increased activity of the renin-angiotensin system Whether or not GRK4 and the angiotensin type 1 receptor (AT
1
R) interact in the aorta is not known. We now report that GRK4 is expressed in vascular smooth muscle cells (VSMCs) of the aorta. Exogenous expression of the GRK4 variant 142V in aortic A10 cells increased AT
1
R protein expression and AT
1
R-mediated increase in intracellular calcium concentration. The increased AT
1
R expression was caused by an increase in AT
1
R mRNA expression via NF-κB, because blockade of NF-κB abolished those effects of GRK4 A142V. As compared with control (vector-transfected) cells, cells expressing 142V had higher NF-kB activity and more NF-kB bound to AT
1
R promoter. The increased AT
1
R expression in cells expressing GRK4 142V was associated with decreased AT
1
R degradation, which was ascribed to the lower AT
1
R phosphorylation. There was direct interaction between GRK4 and AT
1
R in A10 cells which was decreased by GRK4 that could have caused the lower AT
1
R phosphorylation and degradation. The regulation of GRK4 of AT
1
R expression was confirmed in GRK4142V transgenic mice, the AT
1
R expression was higher, while AT
1
R phosphorylation was lower in aorta in GRK4 142V than control mice. Angiotensin II- mediated vasoconstriction was higher in A142V mice. This study provides a mechanism that GRK4, via regulation of arterial AT
1
R expression and function, engaged into the pathogenesis of hypertension.
Abstract 59: Targeted Angiotensin Converting Enzyme Overexpression in Myelomonocytic Cells Prevents Cognitive Decline in Alzheimer’s Disease
Authors: Bernstein Kenneth E , Koronyo Yosef , Salumbides Brenda C , Sheyn Julia , Pelissier Lindsey , Lopes Dahabada H , Shah Kandarp H , Bernstein Ellen A , Fuchs Dieu-Trang , Black Keith L , Shen Xiao Z , Fuchs Sebastien , Koronyo-Hamaoui Maya
Published: March 2022

The cognitive decline in Alzheimer's disease (AD) is associated with elevated brain levels of amyloid β-protein (Aβ), particularly Aβ
1-42
. Angiotensin-converting enzyme (ACE) can enzymatically degrade Aβ
1-42
and its overexpression in myelomonocytic cells enhances their immune function. To examine the effect of targeted ACE overexpression on AD, we crossed ACE
10/10
mice, which over express ACE in myelomonocytic cells, with the double-transgenic APP
SWE
/PS1
ΔE9
mouse model of AD (AD
+
). At 7 months, soluble Aβ
1-42
was reduced by 44% (136 vs 245 pg/mg) and Aβ
1-40
was reduced by 32% (21 vs 31 pg/mg). Plaque burden was reduced by as much as 79% (13 vs 62 10
3
mm
2
) and insoluble Aβ
1-42
by 64% (189 vs 521 pg/mg). There was also a substantial reduction in astrogliosis (49-57% at 7 months, 50% at 13 months). AD
+
ACE
10/10
mice demonstrated less overall brain infiltrating cells, consistent with less AD pathology, though ACE-overexpressing monocytes and macrophages were increasingly abundant surrounding and engulfing Aβ plaque. At 11 and 12 months, AD
+
ACE
10/WT
and AD
+
ACE
10/10
mice were virtually equivalent to non-AD mice in cognitive ability as assessed by maze-based behavioral tests. This study shows that an enhanced immune response, coupled with increased myelomonocytic cell expression of ACE, resulted in essentially complete prevention of the cognitive decline observed in a murine model of AD.
Abstract 604: Renin Independent Ang-(1-12) Production in both the Systemic and Coronary Circulation
Authors: Moniwa Norihito , Varagic Jasmina , Nagata Sayaka , Ahmad Sarfaraz , Simington Steven W , VonCannon Jessica L , Shimamura Yoshinosuke , Hasegawa Koichi , Takizawa Hideki , Ura Nobuyuki , Tanaka Shigemichi , Furuhashi Masato , Yoshida Hideaki , Miura Tetsuji , Ferrario Carlos M
Published: March 2022

Although
angiotensin-(1-12) [Ang-(1-12)] is recognized as an alternate precursor for Ang

II, the biochemical pathways by which the substrate is released from

angiotensinogen (Aogen) are not yet described.

We examined the role of renin on Ang-(1-12) formation from synthetic icosapeptide

[Ang-(1-20)] in the systemic and coronary circulation of Wistar Kyoto rats

(WKY). In experiment 1, rats underwent bilateral

nephrectomy (BNX) or sham operation.

After 48 hours, rats were assigned to sham+saline, sham+Ang-(1-20), or

BNX+Ang-(1-20). Ang-(1-20) (20 nmol/kg/min)

or saline was infused for 15 minutes. At

the end of the study blood was collected for measuring Ang-(1-12), Ang I and

Ang II by RIA. Ang-(1-20) infusion increased

arterial pressure in both sham and BNX compared to sham+saline (P<0.001). Plasma Ang-(1-12), Ang I, and Ang II were significantly

higher in sham+Ang-(1-20) compared to sham+saline. BNX+Ang-(1-20) showed significant increase in

Ang I and a tendency for augmented Ang-(1-12) and Ang II compared to sham+saline

(Figure). In experiment 2, the hearts extracted

from WKY were mounted on Langendorff apparatus.

After recovery, 10 nmol or 100 nmol of Ang-(1-20), or 100 nmol of

Ang-(1-20) with WFML-1 (a rat renin inhibitor) were perfused in the coronary

circulation. Buffer was recirculated

after addition of Ang-(1-20) and WFML-1.

Perfusate was collected at 0, 5, 15, 30, and 60 min for measurement of angiotensins

by RIA. The 100 nmol of Ang-(1-20)

increase Ang-(1-12), Ang I, and Ang II compared to 10 nmol both in the absence

and in the presence of WFML-1 (Figure).

We conclude that Ang-(1-12) is cleaved from Aogen by an enzyme that is

not renin in both systemic and coronary circulation.
Abstract 609: Potential Role Of Adipose Derived Stem Cells In Attenuating Pulmonary Hypertension
Authors: Rathinasabapathy Anandharajan , Shenoy Vinayak , Bruce Erin , Espejo Andrew , Raizada Mohan , Katovich Michael
Published: March 2022

Pulmonary hypertension (PH) is a devastating cardiopulmonary

disorder, which often results in right heart failure. In spite of recent

advancements in the pharmacotherapy of pulmonary hypertension, the mortality

rate has not been significantly reduced. Recently stem cell therapy has gained

more attention for the treatment of cardiopulmonary diseases. Despite the

promising results shown by bone marrow derived stem cells in attenuating right heart

failure in PH, it remains a highly invasive and laborious procedure. On the contrary, adipose derived stem cell

(ADSC) presents a less complicated option. Therefore, we proposed to use ADSC in

attenuating the progression of PH. Monocrotaline

(MCT) at a 50 mg/kg dose was injected to eight-week old male Sprague-Dawley

rats to induce PH. Four weeks post MCT injection; animals developed PH with

elevated right ventricular systolic pressure (RVSP, Control: 28.65±0.40; MCT:

78.06±6.4 mmHg; n=6-8), and right ventricular hypertrophy (RVH, Control;

0.22±0.005; MCT: 0.48±0.054 [ratio, AU]; n=6-8). ADSC (isolated from inguinal fat of healthy male

rat) were characterized using flow cytometry (CD44
+
, CD90
+
,

CD34
-
and CD45
-
) and their differentiation to adipocyte

was confirmed by Oil Red O staining. ADSCs

(1x10
6
; passage 2-4) were injected through the jugular vein, 14 days

post MCT injection. After 2 weeks following

ADSC therapy, RVSP (MCT+ADSC: 55.67±8.99 mmHg; n=6-8) and RVH (MCT+ADSC:

0.32±0.062 [AU]; n=6-8) was significantly reduced. Moreover, the elevated right ventricular end

diastolic pressure (RVEDP) and ±dP/dT associated with PH were attenuated

significantly in ADSC treated MCT animals. An additional group of animals received only

the ADSC conditioned media (CM) from 1x10
6
cells. After 2 weeks of CM administration, RVSP

(MCT+CM: 42.11±4.66 mmHg; n=5) and RVH (MCT+CM: 0.29±0.020 [AU]; n=5) was

significantly reduced. Further, the RVEDP

and ± dP/dT were also significantly reduced in CM treated MCT animals. Collectively, either ADSC or conditioned media

alone can attenuate the progression of PH, which suggest that paracrine-like substances

may be responsible for the beneficial effects observed and may be considered as

a potential therapeutic alternative for the treatment of right heart failure.
Abstract 610: Cardiac and Vascular Hypertrophy Produced by Angiotensin II is Associated with Increases of mTOR/P70S6 Kinases Expression
Authors: zhan shanshan , Gilbert Nicholas L , Sarmiento Joel A
Published: March 2022

It is fairly well-accepted now that Angiotensin II (Ang II)-infusion produces hypertension and cardiac as well as vascular hypertrophy. In cell culture, the mammalian target of rapamycin (mTOR) and its effector kinase (P70S6) have been shown to increase in cardiomyocytes and smooth muscle cells in response to Ang II and have been suggested to contribute to hypertrophy in these two cell types. The goal of this study was to test the Ang II infusion is associated with increased mTOR and P70S6 expression
in vivo
. C57BL/6 mice (3 mo of age) were subjected to either sham (control) surgery or surgically implanted with osmotic containing Ang II (1000ng/mit/kg). In mice implanted with Ang II, systolic blood pressure (SBP) was 134±10 mmHg versus 120±5 in control mice. The heart hypertrophy was indicated by heart weight in Ang II group with 30% heavier than the sham controls (mean ± SEM 165.1 ± 5.5 mg versus 126.4 ± 2.9 mg, P<0.01). Quantitative densitometry evaluation of Western Blot showed the ratio 2447 phosphorylated mTOR / total mTOR was significantly elevated in the heart of AngII group with 54% greater than controls (0.51 ± 0.06 versus 0.33 ± 0.01, P < 0.05); and p389-P70S6/total P70S6 was increased by ~75% in Ang II group versus controls (0.07 ± 0.009 to 0.04 ± 0.002 in Ang II-infused and sham mice, respectively, P<0.05). Similarly, p-mTOR/t-mTOR expression increased by ~17% with Ang II treatment (e.g., p-mTOR/t-mTOR expression was 0.69 ± 0.14 to 0.59 ± 0.14 in Ang II-infused and sham mice, respectively, P<0.05); and p-P70S6 / t-P70S6 in Ang II - infused mice was over 50% greater than controls (0.73 ± 0.15 versus 0.46 ± 0.14, P=0.055).

Conclusion:
mTOR/P70S6 upregulation in the aorta and the heart induced by AngII stimulation indeed happened
in vivo
and indicates mTOR/P70S6 may play important roles in cardiovascular hypertrophy. Also, they may serve as biomarkers or therapeutics indications. The mechanism of mTOR/P70S6 signaling pathway causing hypertrophy should be further explored.
Abstract 611: Interaction of Hypertension and Diabetes in Progressive Nephropathy, Enhanced ER Stress and Mitophagy in Goto Kakizaki Rats
Authors: Wang Zhen , do Carmo Jussara , da Silva Alexandre , Abraham Ralph , Hall John
Published: March 2022

Hypertension is common in patients with diabetes and substantially increases the risk for onset of kidney disease and progression of nephropathy. To investigate the molecular mechanisms by which hypertension and diabetes interact to cause nephropathy, 4 month-old male Goto-Kakizaki (GK) rats, a spontaneous type II diabetic model, were implanted with telemetry probes to measure blood pressure (BP) and heart rate (HR) 24-hrs/day. Hypertension was induced by aorta coarctation (AC) between renal arteries to determine the impact of hypertension plus diabetes in the right kidney (above the coarctation) and normal or slightly reduced blood pressure plus diabetes in the left kidney (below the coarctation). Four weeks after AC, BP above the AC was significantly increased from baseline (111±8 vs. 138±22 mmHg, n=7-9,
p
<0.05). BP measured in the femoral artery showed there was ~25 mmHg pressure gradient across the AC (n=5). Blood glucose (316±60 vs. 251±93 mg/dL) and HR (292±13 vs. 302±23 bpm) levels did not significantly change after AC. Glomerular filtration rate (GFR) in left and right kidneys at 4 weeks after AC were examined by a split bladder method. GFR was increased in the hypertensive-diabetic right kidney compared to the normotensive-diabetic left kidney (1.8±0.2 vs. 0.9±0.2 ml/min,
p
<0.05). Associated with increased GFR, right kidney weight was also increased compared to the left kidney (1.9±0.5 vs. 1.4±0.3 g). Marked renal morphological changes such as thickening of glomerular basement membrane, expansion of mesangial matrix were observed in the right kidney. IHC staining for 4-hydroxynonenal, an indicator of lipid peroxidation and oxidative stress, showed a stronger staining in the right kidney than left kidney. We also observed 17% and 15% increased protein expression on endoplasmic reticulum (ER) stress markers CHOP and GRP 78, as well as 20% increased mitophagy marker Beclin 1 in the right kidney compared to the normotensive left kidney. These results suggest that glomerular hyperfiltration and oxidative stress were induced when hypertension interacted with diabetes. Enhanced ER stress and mitophagy may be two major contributors to amplified renal oxidative stress during the progression of hypertensive-diabetic nephropathy. (NHLBI-PO1 HL51971)
Abstract 615: Progression Of Atherosclerosis Plaque Area In Postmenopausal Women During Controlled Traditional Risk Factors
Authors: Perez Hernan A , Garcia Nestor H , Spence J. David , Armando Luis J
Published: March 2022

The onset of symptoms of coronary heart disease in women are displaced a decade compared with men after menopause, but currently there are reports in which 1 in 4 women is less than 59 years, suggesting that in the pre-menopausal period there is marked progression of vascular disease. Therefore we evaluated the presence of atherosclerotic disease in primary care menopausal women. Additionally, we hypothesized that the strict control of the traditional risk factors is not enough to prevent vascular disease progression.

Methods, post-menopausal women without cardiovascular events from Argentina were evaluated. Framingham risk score with body mass index (FRSbmi) and carotid ultrasound were employed for the evaluation. Carotid total plaque area (TPA) by ultrasound was determined as previously described by Spence et al (Stroke 2002;33;2916-2922). Additionally, LDL cholesterol (LDL), triglycerides (Trig) and HbA1c were measured. All determinations were done on day 1 and after 8 months of treatment. Only patients who achieved all the therapeutic goals (blood pressure (BP)<130/80mmHg, LDL<100mg/dl, Trig<140mg/dl, HbA1c5mm2, stable group, TPA changed 5mm2. In the presence of controlled of traditional risk factors, 50% of the population continued to progressive atherosclerotic disease (Table).

Conclusion, these results indicate that in the presence of controlled traditional risk factors, atherosclerosis may progress in some patients, including in menopausal women. Additional promoters of atherosclerosis should be evaluated in these progressive patients or possibly more intensive treatment should be used in such patients.
Abstract 62: Novel role of Aminopeptidase A enzyme in Ang-(1-7) metabolism Post Myocardial Infarction
Authors: Alghamri Mahmoud , Grobe Nadja , Elased Khlalid , Meszaros Gary , Luther Dan , Morris Mariana
Published: March 2022

Aminopeptidase A (APA) is a metalloprotease that is activated in cardiovascular diseases. Ang-(1-7) has been shown to have beneficial effects in myocardial injury. The aims of this study are to (1) Test the role of APA in cardiac pathology using the myocardial infarction (MI) model, (2) Investigate the role of APA in Ang-(1-7) metabolism (3) Explore the effect of APA inhibition on Ang-(1-7) homeostasis. APA activity was measured using mass spectrometry (MS) based in vitro and in situ enzyme assays. For the in vitro assay, heart homogenates were incubated with 50 μM Ang II in assay buffer and for absolute quantitation stable-isotope labeled internal standards were added. In the in situ MS imaging, frozen myocardial sections were incubated with 100 μM Ang II for 10 min and the resultant peptides were assessed on the tissue sections. For kinetic studies and Km determination, recombinant APA was incubated with Ang II and Ang-(1-7) in concentrations ranging from 1-100 μM. Results from the in vitro MS enzyme assays showed that myocardial APA activity was significantly increased after 24 hrs MI compared to sham (Ang III=0.82±0.02 vs. 0.32± 0.02 ρmol/min/μg in MI vs sham, respectively, p<0.01). The in situ MS imaging results confirmed that Ang III intensity was higher in MI heart sections compared to sham (96325±214 for MI and 1041±75 AU for Sham, p<0.05). Both MS enzyme assays identified a new peptide Ang-(2-7), m/z 784, which accumulated in the MI compared to Sham (136.96± 6.5 vs 78.94±3 AU in MI and Sham, respectively, p<0.05). Inhibition of APA with 4-Amino-phosphono-butyric acid significantly decreased formation of Ang-(2-7) from Ang-(1-7) and Ang III from Ang II by 98± 0.7% and 82±2.6% (p<0.05), respectively, indicating that APA is the responsible enzyme for the myocardial formation of both peptides. Interestingly, APA exhibited a higher substrate affinity for Ang-(1-7) compared to Ang II (Km for Ang II=14.32±1.6 vs Km for Ang-(1-7) =6.34± 1.12 μM, p<0.05). In conclusion, we have shown for the first time that APA is responsible for Ang-(1-7) degradation to Ang-(2-7). An upregulation of this pathway in myocardial injury may deprive the heart of cardioprotective Ang-(1-7). Thus, APA can be a potentially novel therapeutic target for management of myocardial remodeling after MI.
Abstract 626: Caveolin-1 (CAV1) Levels Determine the Cell Surface Expression of the Dopamine D1-like Receptors
Authors: Bigler Wang Dora , Van Sciver Robert E , Gildea John J , Felder Robin A
Published: March 2022

In human renal proximal tubule cells, the dopamine-1 receptor (D1R) increases cAMP production and negatively regulates sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein coupled receptor kinase type 4 (GRK4), which hyperphosphorylates and decreases D1R function, leading to sodium retention. We have previously shown that knockdown of CAV1 in proximal tubule cells impairs sodium transport. In this report we show that CAV1 reduction, knockdown or knockout leads to decreased dopamine D1-like receptor binding at the proximal tubule cell surface. Dopamine D1-like brush border and cell surface binding was measured using Bodipy-FL conjugated
SKF83566
. WT and Caveolin 1 knockout (CAV1KO) kidney cortex brush border membrane isolation was performed by magnetic lotus tetragonolobus agglutinin affinity purification and fluorescent
SKF83566
binding by microplate fluorometry. CAV1KO had a 35.8±3.3% decrease in binding vs WT (p<0.01, n=6 per group, WT level 18,492 RFU above background). In hTERT immortalized human renal proximal tubule cell lines, lentiviral shRNAi CAV1 stable knockdown cell lines had a 76.4±2.5%, p<0.001 n=4, reduction in CAV1 expression by western blot. Cell surface D1-like receptor binding was reduced by 34.0±1.3% (p<0.001, n=4) in the CAV1 knockdown cell lines (scrambled control levels 117193 RFU). We previously have shown that CAV1 expression is dramatically reduced in the hypertensive SHR proximal tubule cell line vs the normotensive WKY proximal tubule cell line. The SHR cell line had a 57.5±1.2% (p<0.001, n=7) reduction in D1-like receptor binding (WKY 116114 RFU). Re-expression of rat CAV1 alpha or CAV1 beta isoform in the SHR cell line by stable lentiviral infection increased binding by 51.6±2.1%, and 63.3±1.6% respectively (p<0.01, n=14). Sodium transport was decreased by CAV1 alpha and CAV1 beta expression in SHR cell lines. The average number of domes per 10x objective fields of view was 9.5±1.6 in SHR cell, 4.5±0.3 in SHR CAV1alpha (p<0.05, n=4 vs SHR) and in SHR CAV1beta 5.0±0.3 (p<0.05, n=4 vs SHR). In mouse, rat, and human renal proximal tubule cells, CAV1 is necessary for dopamine D1-like receptor cell surface localization and for normal dopaminergic inhibition of sodium transport.
Abstract 628: Early Life Stress Exacerbates The Adrenergic Receptor Subtypes Mrna Expression In The Kidney
Authors: Loria Analia S , Pollock Jennifer S
Published: March 2022

Maternal separation (MS) in rodents is a model of human chronic behavioral stress during early life. Previously we have shown that renal sympathetic system is sensitized in adult MS rats. Accordingly, we found that reduced glomerular filtration rate in intact MS rats was normalized after bilateral renal denervation (DnX). The aims of this study were to test the mRNA expression of adrenergic receptor (AR) subtypes in intact and DnX renal cortex and renal vessels. Also, to test the levels of mediators related to the alpha (1,4,5-trisphosphate, IP3) and beta (adenosine-3′,5′-cyclic monophosphate, cAMP) AR signaling pathways. MS was performed in WKY by separating approx half of the male pups from the dam 3 hr a day from days 2-14 of life. Renal tissue from 12 weeks old rats was harvested and snapped frozen. RT-PCR was performed using primers for ARs and IP3 receptor 1 and 2 and mRNA levels. cAMP was determined in renal cortex homogenates by ELISA. IP3 receptor mRNA expression was not different between MS and control rats in intact cortex, Dnx cortex or kidney vessels. cAMP was increased in intact renal cortex of MS rats compared to control (2.37±0.23 vs. 1.55±0.0.24 pmol/mg protein, respectively, p<0.05). Relative mRNA expression (2
-
ΔΔ
ct
) of α1a, α1b, α1d and α2b were not different in intact cortex from control and MS rats, while Dnx did not have any significant effect. However, α2c AR was increased in intact renal cortex from MS rats compared to control rats (4.5±1.2 vs. 1.8±0.3 2
-
ΔΔ
ct
, p<0.05). DnX significantly reduced α2c mRNA expression in renal cortex from MS and control rats (0.9±0.2 and 0.5±0.1 2
-
ΔΔ
ct
, p<0.05 vs. intact cortex). Relative mRNA expression of α1a, α1b, α1d, α2b and α2c were significantly increased in renal vessels tissue from MS rats compared to control rats (p<0.05).

We conclude that enhanced renal mRNA expression of ARs, especially α2c, may be a key mechanisms contributing to the lower glomerular filtration capacity in MS rats. Distinctive pharmacodynamic properties of α2c ARs suggest that they may be attractive targets for therapeutic intervention in case of exposition to early life stress.
Abstract 632: Does Cardiac Ang-(1-12) Have A Role In Sex-specific Heart Disease?
Authors: Nagata Sayaka , Varagic Jasmina , Simington Stephen W , Ahmad Sarfaraz , VonCannon Jessica L , Wang Hao , Kon Neal D , Dell'Italia Louis J , Ferrario Carlos M , Groban Leanne
Published: March 2022

Cardiovascular diseases affect women and men differently and large clinical trials suggest ACE inhibitors may be less effective in women than men receiving treatment for hypertension and heart failure. The discovery that chymase forms Ang II directly from Ang-(1-12) independent of ACE and renin in human hearts, prompted the characterization of sex differences in the cardiac expression of Ang-(1-12) and chymase in the heart of subjects undergoing cardiac surgery. Therefore, the objective of this pilot study is to determine whether the expression and activity of Ang-(1-12) and chymase in the human heart are associated with cardiovascular phenotypes in a sex-specific manner. The portions of left (LA) and right (RA) atrial appendages were obtained from male (LA =13 and RA=9) and female (LA=3 and RA=5) patients undergoing heart surgery. A high affinity purified antibody directed to the COOH-terminus of the full length of human Ang-(1-12) was used to detect immunoreactive Ang-(1-12) in all samples (LA: male=13 and female=3; RA: male=9 and female=5) while control sections were treated with the primary antibody preincubated with 0.115 μM of the Ang-(1-12) peptide to which the antibody was directed. Quantitative analysis was performed with Image J software. Native plasma membranes from LA (males=7 and females=3) and RA (males=7 and females=3) were prepared for chymase activity determination by HPLC. Quantitative analysis of Ang-(1-12) expression revealed significantly higher Ang-(1-12) expression in LA appendages of females (Intensity: 61.41±13.43 units, P=0.04) versus males (29.36±6.07 units) while no sex differences were observed in the RA. Chymase activity also tended to be lower in the LA of females (40.22±9.80 fmol/mg/min) vs. males (57.08±5.48), but values were similar between sexes in the RA. These sex-specific differences in the noncanonical RAS may provide important insight into sex-dependent pathophysiology underlying heart disease in women and in therapeutic decisions in the future.
Abstract 637: Angiotensin Receptor Blockade Ameliorates the Hypertension Associated with Acute Glucagon-like Peptide-1 Receptor Activation
Authors: Rodriguez Ruben , Lee Andrew , Moreno Meagan , Vazquez Guillermo , Duval Ruilova Steven , Nishiyama Akira , Ortiz Rudy M
Published: March 2022

Insulin resistance (IR) is associated with elevated renin-angiotensin system (RAS), impaired urinary Na
+
excretion (U
Na
V), and high blood pressure. RAS blockade lowers blood pressure, decreases IR and fasting plasma glucose (FPG). Glucagon-like peptide 1 (GLP-1), has been reported to have natriuretic effects as well as blood glucose lowering effects, however, these findings are still controversial. To assess the contributions of GLP-1 receptor and angiotensin receptor type 1(AT1) activation on renal Na
+
handling, systolic blood pressure (SBP) and FPG, metabolic measurements were taken at days 0, 2, 7, and every week thereafter for six weeks, SBP was measured daily using radio telemetry in five groups of rats: 1) untreated, lean LETO (n=7), 2) untreated, obese OLETF (n=5), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan /kg/d; n=4), 4) OLETF + GLP-1 mimetic (Ex; 10 ug exenatide/kg/d; n=7), and 5) OLETF + ARB + Exenatide (combo; n=6). FPG increased (101 ± 4 vs 131 ± 3 mg/dL) in OLETF compared to LETO, and ARB treatment decreased it (120 ± 2 mg/dL); however, the changes in Ex and combo groups were not significant. OLETF rats exhibited elevated SBP at the onset of the study compared to LETO (119 ± 1 vs. 130 ± 1 mmHg). U
Na
V decreased (1.07 ± 0.07 vs 0.43 ± 0.14 & 0.31 ± 0.11 mmol/day) in Ex and combo groups on day 2 compared to OLETF, and the decrease in U
Na
V in Ex was associated with increased (132 ± 1 vs 142 ± 1 mmHg) SBP. However, reduced U
Na
V in combo group was associated with reduced (114 ± 3 mmHg) SBP suggesting that activation of AT1 has a greater contribution to the insulin resistance-associated increase in SBP than the impaired U
Na
V. After this initial increase in SBP in Ex, it decreased to a nadir (124 ± 2 mmHg) at day 11 of treatment before increasing to 130 for the duration of the study. After six weeks of treatment U
Na
V increased (1.02 ± 0.05 vs 1.35 ± 0.09 mmol/day) only in combo group compared to OLETF. The results suggest that chronic GLP-1 receptor activation acutely increases SBP by decreasing U
Na
V, but co-treatment with an ARB abolishes this effect. These results also suggest that despite the lack of improved FBG, chronic GLP-1 receptor activation may protect against the development of high blood pressure associated with insulin resistance independent of increased U
Na
V.
Abstract 640: Role Of Endothelial No Synthase Expression On Endothelial Dysfunction In Hypertensive Patients
Authors: Danesh Shahab M , Solow Elizabeth B , Arbique Debbie , Wang Zhongyun , Onwuegbuchu Erica , Mineo Chieko , Vongpatanasin Wanpen
Published: March 2022

Previous studies have implicated impairment

in the expression of endothelial NO synthase (eNOS) and the S1177 phosphorylated

form of eNOS in mediating endothelial dysfunction in the spontaneously

hypertensive rats. However, the relevance of this observation in human

hypertension remains unknown. Accordingly, we assessed endothelial function in 19

patients with untreated essential hypertension (HTN) and 18 age-matched

normotensive controls (NT), using noninvasive flow mediated vasodilation (FMD)

in the brachial artery. We also assessed endothelial cell (EC) protein

expression of eNOS and S1177 phosphorylated eNOS using immunofluorescence staining of the ECs extracted via J wire

insertion into antecubital veins. We found that FMD was significantly reduced in

the HTN versus the NT group (6.67±0.53 % vs. 9.16±0.98

%, p < 0.05). Despite impairment in endothelial function, we found that

protein expression of total eNOS and S1177 phosphorylated eNOS were not

significantly different between the HTN and NT groups (0.46±0.07 vs.

0.38±0.05 and ±0.05 vs. 0.36±0.08, p = NS,

respectively). In conclusion, our study suggests that impairment in the endothelial

vasodilator function is independent of

eNOS or S1177 phosphorylation of eNOS protein

expression in human hypertension. Further studies are needed to clarify the

molecular basis of endothelial dysfunction in hypertensive patients.
Abstract 642: NOS-dependent Increase In Splanchnic Blood Flow Causes The Hypotensive Response To 24-hour 5-HT Infusion
Authors: Seitz Bridget M , Watts Stephanie W
Published: March 2022

Long-term serotonin (5-hydroxytryptamine, 5-HT) infusion produces a sustained reduction in mean arterial pressure (MAP) and elicits a decrease in total peripheral resistance (TPR) through a mechanism dependent on nitric oxide synthase (NOS) in rats. We sought to determine if the reduction in TPR induced by 5-HT is caused by NOS-dependent relaxation of vessels to all major organs of the body or selective action on a single vascular bed. Alzet mini-pumps containing vehicle (saline) or 5-HT (25 μg/kg/min) were implanted in male Sprague Dawley rats. One-week prior to pump implantation, rats were either: (1) switched to drinking water containing the NOS inhibitor N-nitro-L-arginine (L-NNA; 0.5 g/L), or (2) maintained on tap water. 5-HT reduced MAP after 24 hours (veh = 87±3, 5-HT = 75±2 mm Hg; p<0.05), which was prevented by L-NNA (L-NNA/5-HT= 115 ±10 mm Hg; p<0.05). After 24 hours of 5-HT infusion, yellow-green fluorescent microspheres, used to determine blood flow (300, 000 spheres; size 15μm), were introduced into the left ventricle of the heart. Microspheres were recovered in arterial reference blood sample and organs using sedimentation through centrifugation. The fluorescence intensity of the dye extracted from the microspheres was measured by spectrophotometery. 5-HT significantly decreased resistance to the spleen (48%), small intestine (35%), and adrenal (70%) compared to vehicle-treated animals, and this decrease was abolished by L-NNA. By contrast, resistance to the ear (175%) was increased by 5-HT. Resistance to other organs (brain, liver, lungs, kidneys and muscle) did not show a statistically significant reduction by 5-HT. Collectively, these findings underscore the importance of the splanchnic circulation and NOS in 5-HT-induced reduction in blood pressure.
Abstract 645: Atorvastatin and Sildenafil Reduce Oxidative Stress, MMP-2 And MMP-9 Levels, TGF-beta Expression and Prevent Vascular Remodeling in 2K1C-hypertension
Authors: Guimaraes Danielle A , Rizzi Elen , Ceron Carla S , Oliveira Alisson M , Gerlach Raquel F , Tanus-Santos Jose
Published: March 2022

Hypertension-induced vascular remodeling is associated with oxidative stress, and matrix metalloproteinase (MMP) and TGF (transforming growth factor)-beta up-regulation. Atorvastatin (ATORVA) and sildenafil (SILD) exert pleiotropic effects that may result in cardiovascular protection. We evaluated the effects of ATORVA and SILD alone or in association on oxidative stress, MMPs and TGF-beta up-regulation, and vascular hypertrophy induced by 2 kidney-1 clip (2K1C) hypertension. Sham and 2K1C rats were orally treated with vehicle, ATORVA (50 mg/kg), SILD (45 mg/kg) or both for 8 weeks. ATORVA, SILD, or both drugs exerted antihypertensive effects (systolic blood pressure: 148±7, 156±5, and 138±4 mmHg, respectively,
vs.
207±4 mmHg in 2K1C untreated rats; P<0.05). All treatments prevented the increases in the aortic cross-sectional area and media/lumen ratio in 2K1C rats (P<0.05). Hypertension increased NADPH oxidase activity when compared with control groups and the treatments reduced oxidative stress. Aortas from 2K1C rats showed higher MMP-2 levels when compared with sham (0.399±0.06
vs.
0.173±0.05 A.U.) and all treatments attenuated 2K1C hypertension-induced increases in MMP-2 levels (ATORVA: 0.191±0.04; SILD: 0.136±0.05; ATORVA+SILD: 0.173±0.03; P<0.05). However, these drugs had no
in vitro
effects on human recombinant MMP-2 activity (P>0.05). Moreover, 2K1C rats showed higher MMP-9 (12.64±0.86
vs.
8.70±0.92) and TGF-beta expression (14.25±0.97
vs.
9.07±1.67) compared with sham groups. All treatments prevented 2K1C hypertension-induced increases in these pro-fibrotic factors. Treatment with ATORVA or SILD, or both, exerted antihypertensive effects and prevented 2K1C hypertension-induced vascular remodeling through antioxidant and anti-fibrotic mechanisms. Our results suggest that ATORVA and SILD may prevent the vascular alterations of hypertension.
Abstract 67: Anti-aging Gene Klotho Deficiency Causes Hypertension Via Upregulation of CYP11B2 Expression and Aldosterone Synthesis
Authors: Zhou Xiaoli , Lei Han , Sun Zhongjie
Published: March 2022

Background -
Klotho (KL) is a recently discovered anti-aging gene. Genetic mutation of KL expedites the aging process and shortens lifespan while overexpression of KL slows down the aging process and extends lifespan. The purpose of this experiment is to assess if KL deficiency affects blood pressure (BP).

Methods & Results -
KL mutant heterozygeous (+/-) mice showed significant and persistent elevation of BP, demonstrating for the first time that half klotho deficiency causes hypertension. It is noticed that plasma level of aldosterone was elevated and that protein expression of CYP11B2 (key enzyme of aldosterone synthesis) in adrenal cortex was increased in KL(+/-) mice, indicating that KL deficiency upregulates aldosterone synthesis. Blockade of aldosterone receptors by eplerenone (6 mg/kg/day) decreased hypertension of KL(+/-) mice to the control level within one week, suggesting that upregulation of aldosterone levels may play a critical role in KL deficiency-induced hypertension. Indeed, the aldosterone signaling (SGK1, NCC and ATP-β) were increased in kidneys of KL(+/-) mice. In addition, half KL deficiency increased renal inflammation as evidenced by upregulated inflammatory factors (IL-6, TNFα, osteopontin and MCP-1) and increased infiltration of T cells and macrophages in kidneys of KL(+/-) mice. Half KL deficiency led to renal functional impairment (increased plasma creatinine and urea and urine albumin) and structural damage (glomerulus collapse, tubule fibrosis). Blockade of aldosterone receptors attenuated KL deficiency-induced renal inflammation and damage.

Conclusions -
KL is essential to the maintenance of normal BP. KL deficiency caused hypertension and kidney damage
via
upregulating aldosterone levels and consequently increasing inflammation and SGK1 and NCC signaling in kidneys.
Abstract 68: Mineralocorticoid Receptor Activation Leads to Ocular Choroidal Vessels Dilation and Leakage in Rats and Human Central Serous Chorioretinopathy
Authors: Jaisser Frederic , Zhao Min , Farman Nicolette , Behar-Cohen Francine
Published: March 2022

We have shown that several cell types of the rat retina express the mineralocorticoid receptor (MR) and the MR-protecting enzyme HSD2 (Zhao et al, FASEB J 2010, 24:3405), raising the question of the role of the MR in normal and diseased retina. We now questionned the role of MR in the choroid vessels lying in the outermost posterior part of the eye. Intravitreous injection of the MR ligand aldosterone in rat eye induced, 24 hrs later, dilation of choroid vessels and fluid leakage, through specific MR activation. Corticosterone (at high doses, not low doses) elicited the same effect, presumably via MR occupancy. We identified an underlying mechanism: aldosterone up-regulates the endothelial vasodilatory potassium channel KCa2.3; its blockade(by intravitreous apamine) prevents aldosterone-induced thickening.

We seached whether human retinal diseases may recapitulate some of the features of MR overactivation. Central serous chorioretinopathy (CSCR) is a vision-threatening eye disease affecting young stressed men, with unknown pathogeny and no validated treatment. Subretinal fluid accumulation originates from dilation and leakage of choroid vessels underneath the retina. Recurrence or chronic evolution may occur, leading eventually to vision loss. Based on aggravation of CSCR by glucocorticoids , on identification of systemic and/or locoregional glucocorticoids as known risk factors, and on the recognized MR affinity for glucocorticoids, we hypothesized that inappropriate/excessive occupancy of the MR by glucocorticoids may occur in the choroid of CSCR patients. Four patients with chronic non-resolved CSCR (and reduction of visual acuity) received low doses of oral eplerenone for 5 weeks. Retinal detachment and choroidal vasodilation were rapidly (1- 2 weeks) resolved; this impressive recovery was associated with improved visual acuity. The benefit was maintained after eplerenone withdrawal. Controlled clinical trials are now required to assess this novel therapeutic approach.

In conclusion, our results identify MR signaling as a novel pathway controlling choroidal vascular bed relaxation and provide a pathogenic link with human CSCR, leading to proposal of short-term MR blockade to reverse the choroid vasculopathy and improve vision.
Abstract 72: Redox-sensitive Transcriptional Regulation of Renal Dopamine D1 Receptor Function during Hypertension
Authors: Banday Anees A , Lokhandwala Mustafa F
Published: March 2022

Oxidative stress plays an important role in the development of hypertension. We and others have shown that renal dopamine D1 receptor (D1R), a major contributor to sodium homeostasis and BP regulation, is negatively regulated by oxidative stress. It is reported that antioxidants can reduce BP and redox-sensitive transcription factor Nrf2 could provide important clues for antihypertensive properties of antioxidants. Here in we investigated the signaling molecules responsible for redox-sensitive renal proximal tubular D1R dysfunction and how the induction of renal specific Nrf2, in response to antioxidants, will protect D1R function and reduce BP. Wild type (WT) and renal proximal tubular specific Nrf2 knockout (KO) mice were treated with 10 mM L-buthionine-sulfoximine (BSO, a pro-oxidant) without and with sulforaphane (S, a polyphenol antioxidant; 5 μM daily by gavage) for 5 wks. In WT mice, BSO treatment increased oxidative stress and BP (mm Hg, WT: 104 ± 3, WT-BSO: 125 ± 5), reduced renal tubular D1R expression and attenuated
SKF38393
(a D1R agonist)-induced Na/K-ATPase inhibition and sodium excretion. BSO also activated transcription factors activator protein (AP) 1 and SP3. However, the onset and severity of hypertension (BP, Nrf2-KO: 101 ± 3, Nrf2-KO-BSO: 137 ± 4) as well as oxidative stress, activation of AP1 and SP3 and D1R dysfunction were more robust in BSO-treated Nrf2 KO compared to WT mice. More importantly, sulforaphane activated Nrf2, reduced oxidative stress, normalized AP1 and SP3 activation, rescued DIR function and lowered BP (WT-S: 97 ± 4, WT-BSO+S: 109 ± 6) in BSO-treated WT mice but failed to normalize renal D1R function or reduce BP (Nrf2-KO-S: 99 ± 3, Nrf2-KO-BSO+S: 131 ± 4) in Nrf2 KO mice. Further experiments in human proximal tubular cultures revealed that BSO via AP1 activated SP3 which in turn transcriptionally down-regulated D1R expression and sulforaphane, via Nrf2 activation, abolished BSO-induced AP1 and SP3 activation and protected D1R function. In conclusion, oxidative stress via AP1-SP3 pathway down-regulates D1R expression and function which leads to sodium retention and hypertension. Antioxidants, via Nrf2 activation, reduce oxidative stress and normalize AP1-SP3 signaling and D1R function which reduces BP.