bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P224: Zamicastat Prevents the Deterioration of Cardiometabolic and Inflammatory Biomarkers in a Genetic Model of Salt-sensitive Hypertension
Authors: Igreja Bruno , Pires Nuno , Wright Lyndon C , Soares-da-Silva Patrício
Published: March 2022

The Dahl salt-sensitive (Dahl/SS) rat, a genetic model of salt-sensitive hypertension and heart failure, develops left ventricle (LV) hypertrophy after five to six weeks and cardiac failure with LV dilation and contractile dysfunction after 10 to 12 weeks of high-salt (HS) intake. The aim of the present study was to evaluate the effect of zamicastat, a selective peripheral dopamine β-hydroxylase inhibitor, on cardiometabolic and inflammatory biomarkers in Dahl/SS during chronic HS intake.

Eight week-old Dahl/SS rats were randomized into three groups; two groups received for 10 weeks a HS (8% NaCl) diet, one of which received 30 mg/kg/day zamicastat (HS+ZAMI); a third cohort received normal-salt diet (NS-0.3% NaCl) and served as controls. In the last week of treatment, 3 out of 10 animals from the HS group and 1 out of 10 from HS+ZAMI group were found dead. All animals from the NS group survived (n=8). Rats in the HS and HS+ZAMI groups showed heart and kidney hypertrophy as indicated by higher heart/body weight ratios (3.86±0.05 and 3.63±0.09 vs 2.96±0.05 mg/g) and kidney/body weight ratios (5.23±0.29 and 4.80±0.24 vs 3.41±0.06 mg/g), as compared to the NS group. A multiplex inflammatory cytokine assay was used to profile expression of 23 inflammatory mediators. Plasma levels of IL-1β, IL-4, IL-5, IL-7 and GM-CSF were significantly increased in the HS, but not in the HS-ZAMI group. Plasma levels of monocyte chemoattractant protein (MCP)-1 were higher in the HS and HS+ZAMI than in NS group (105.0±10.4 and 85.2±5.9 vs 67.6±2.8 pg/ml). The HS group, but not HS+ZAMI, showed hypercholesterolemia (4.45±0.23 and 3.72±0.37 vs 3.07±0.22 mmol/l). Both HS and HS+ZAMI cohorts had reduced levels of free fatty acids (0.23±0.02 and 0.23±0.02 vs 0.35±0.02 mmol/l) and normal levels of triglycerides in plasma (1.87±0.29 and 1.69±0.18 vs 1.28±0.15 mmol/l). Insulin plasma levels in HS and HS+ZAMI groups were lower than in NS group (1.45±0.26 and 1.90±0.22 vs 4.03±0.40 ng/ml), but the glucose levels were similar in all three groups (162±5 and 168±7 vs 150±7 mg/dl).

In conclusion, chronic HS intake deteriorates several cardiometabolic and inflammatory biomarkers in Dahl/SS rats, which can be prevented by dopamine β-hydroxylase inhibition with zamicastat.
Abstract P233: Sodium Glucose Transporter Type-2 (SGLT-2) Inhibitor, Empagliflozin, Improves Cardiovascular Outcomes in Female Diabetic db/db Mice Independent of Blood Pressure Reduction
Authors: Sowers James , Aroor Annayya , Jia Guanghong , Nistala Ravi , Garro Mona , Meininger Gerald , Martinez-Lemus Luis , Mayoux Eric , Whaley-Connell Adam , DeMarco Vincent G
Published: March 2022

Dysglycemia, proteinuria, vascular stiffness, diastolic dysfunction (DD) and hypertension are abnormalities seen more frequently in the obese, diabetic population. SGLT-2 inhibitors (SGLT-2i), which increase urinary glucose/sodium excretion to lower HbA1c and blood pressure (BP), are emerging as unique diabetes therapies. We examined whether the SGLT-2i, empagliflozin (EMPA), ameliorates hypertension, dysglycemia, proteinuria, aortic stiffness and DD in obese/diabetic female db/db mice. Eleven week old mice were fed a diet with or without EMPA (10mg/kg/day) for 5 weeks. In vivo blood pressure (telemetry), diastolic function (echo), aortic stiffness (pulse wave velocity, PWV), proteinuria, renal resistivity (echo) and HbA1c were evaluated. Db/db had elevated BP that was not affected by SGLT2i. HbA1c, proteinuria and the renal resistivity index (RI) were elevated (P<0.001) in control (Db) mice vs treated mice (Db-EMPA) and lean control mice. Db exhibited DD that was ameliorated by SGLT2i as indicated by reduced LV filling pressure (<E/E’) and improved septal wall motion (E’/A’ ratio, not shown). Elevated PWV and aortic endothelial cell stiffness in Db-C were abrogated with SGLT2i. These results show that SGLT2i confers BP-independent cardiovascular and renal protective effects in obese diabetic female mice.
Abstract P236: Alginic Acid May Not Play a Major Role in the Mechanism of Alleviating Hypertension by Dietary Saccharina Japonica in 2-kidney, 1-clip Renovascular Hypertensive Rats
Authors: Segawa Yukiko , Maruyama Saki , Hashimoto Hiroko , Osera Tomoko , Kurihara Nobutaka
Published: March 2022

Objective:
Saccharina japonica (SJ), one of brown algae, is cultivated or grows wild in East Asia. The extract ‘dashi’ is used for soup stock in Japan. The intake of SJ was reported to decrease blood pressure (BP) in 2-kidney, 1-clip renovascular hypertensive (2K1C) rats in our previous study, as well as in spontaneously hypertensive rats (SHR) in other studies. Some investigators suggested that a major mechanism of reducing BP by dietary SJ in SHR includes alginic acid (AA). However, in our preliminary study it was not confirmed in 2K1C model, because we observed that dashi containing AA as much as 5% of what the original SJ contained alleviated the hypertension, too. To detect how AA in SJ contributes to the mechanism, we observed BP in 2K1C rats fed a diet containing SJ or extracts from SJ.

Methods:
Male Sprague-Dawley rats (6 wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, the rats started receiving a control diet (C) or a diet containing 5% (w/w) SJ (S), dashi extracted from the same amount of SJ in a correct method (D), extract from SJ equal in amount by boiling for a long time, in which AA was much eluted (B) or the SJ leftover after dashi was extracted (L) for 6 weeks. The systolic BP (SBP) was measured by a tail-cuff method every week. At the end, mean arterial BP (MAP) was measured in each rat under anesthesia. After euthanasia, the aortas were collected for extracting mRNA and protein. Endothelial nitric oxide synthase (eNOS) mRNA expression (eNOS-M) was evaluated in aortas by reverse transcriptase-polymerase chain reaction. eNOS and phosphorylated eNOS protein expression (eNOS-Ps), was determined in aortas by western blot analysis.

Results:
Six weeks after the surgery, SBP was significantly higher in 2K1C-C than in SHAM-C (182 ± 6 vs 126 ± 5 mmHg, P < 0.001). In 2K1C-S, -D, -B and -L (145 ± 4, 148 ± 4, 133 ± 3 and 146 ± 4 mmHg), SBP was significantly lower than that in 2K1C-C (P < 0.001, each). At the end of the protocol, MAP showed similar results to SBP. No significant differences were found in eNOS-M between each groups. eNOS-Ps were enhanced in 2K1C compared to SHAM (P < 0.05), but showed no significant differences by diet.

Conclusion:
The role of AA and eNOS may be limited in the mechanism of alleviating hypertension by dietary SJ in 2K1C.
Abstract P239: Could Uric Acid Levels Predict Success In Chronic Weight Loss?
Authors: Masuo Kazuko
Published: March 2022

Background:
Obese individuals have higher prevalence of hyperuricemia (hyperUA) and with weight loss (WL) improved hyperUA. We previously reported that serum uric acid levels (UA) associated plasma norepinephrine (NE) could predict future weight gain and BP elevation, and that WL program combining diet + exercise was most effective on UA, WL and BP reductions, although diet had stronger effects on UA and BP reduction compared to exercise.

Objective:
In this study, we examined whether lower UA levels would predict future success and maintenance of chronic WL compared to higher UA levels in a 2 yr WL regimen with diet and exercise.

Methods:
154 overweight/obese men (BMI>25 kg/m2) consisting of 89 normotensive and 65 untreated mild hypertensives were enrolled in the WL program with diet + exercise. The subjects had BP, BMI, Fat-mass, UA, plasma NE, insulin, glucose, HOMA-IR measured at entry, 6, 12, and 24 months. WL was defined as ≥10% WL.

Results:
95 subjects succeeded significant WL at 24 month including 34 subjects in WL Maintenance group (WL was observed at both 6 and 24 m), and 61 subjects with Slow WL (WL was observed at only 24 m). At entry, serum UA and plasma NE in WL group (WL maintenance + Slow WL) were slightly lower compared to those in non-WL groups (WL<10% at 24 m). Especially the slow WL group had significantly lower UA at entry compared to the non-WL group and WL maintenance group. Plasma UA and NE levels in the WL group were significantly lower than the non-WL group. Percent (%) reductions of UA and NE were greater in WL group than non-WL group. In all subjects, baseline UA and % changes of UA correlated with % changes in BMI over 24 m (R=0.205, P<0.05; R=0.230, P<0.05, respectively). Baseline UA correlated with % changes in UA, changes in Fat-mass, and changes in BMI (R=0.210, P<0.05; R=0.197, P<0.05; R=0.185, P<0.05, respectively). Plasma NE at entry correlated with changes in UA and % changes in UA in all subjects (R=0.263, P<0.05; R=0.412, P<0.01). BP reductions at 24 m correlated with WL and baseline UA in all subjects (R=0.345, P<0.05; R=0.247, P<0.05) respectively).

Conclusions:
Serum UA and plasma NE levels may predict success in chronic WL with WL regimens, and may predict BP reduction associated with WL.
Abstract P243: A Maternal High Salt Diet During Pregnancy and Lactation Affects Offspring Cardiac Function
Authors: Kagota Satomi , Maruyama Kana , Van Vliet Bruce N , Wakuda Hirokazu , Shinozuka Kazumasa
Published: March 2022

The maternal environment during pregnancy and lactation has important effects on the offspring’s cardiovascular system. For example, changes in offspring blood pressure and vascular structure have been reported to occur in response to a maternal high salt intake during pregnancy and lactation. However, it remains unclear whether a maternal high salt intake may affect cardiac function in offspring. We previously demonstrated that high salt diet causes aggravation of hypertension in young spontaneously hypertensive rats (SHR). Therefore, in the present study we investigated the influence of exposure to a maternal high salt diet during gestation and lactation on offspring cardiac function in SHR.

SHR dams were fed either a high salt diet (4% NaCl) or a control (0.3% NaCl) diet. After weaning, the offspring were fed the control diet for 8 weeks. Systolic blood pressure and heart rate of the offspring were measured without heating using a photoplethysmographic tail-cuff system at 11 weeks of age. Indices of both left ventricular (LV) systolic and diastolic function (LV developed pressure [LVDP], and maximum rate of LV decline [-dP/dt]) and coronary flow were determined in 12 week old offspring using a Langendorff isolated perfused working heart system.

Offspring of the high-salt diet-fed dams (High salt) had lower blood pressure, heart rate, and indices of both LV systolic and diastolic function, compared with offspring of the control diet-fed dams (control) (Fig.). There were no significant differences in coronary flow or heart weight between the two groups. These results suggest that a maternal high salt intake is a predisposing factor for disturbance of cardiac function in offspring.
Abstract P245: High-salt Diet Induces Catabolic Urea Formation and Muscle Wasting to Enable Renal Salt Concentration
Authors: Kitada Kento , Zhang Yahua , Neubert Patrick , Pedchenko Tetyana , Lantier Louise , Wasserman David , Luft Friedrich C , Titze Jens
Published: March 2022

Background:
We showed previously that a high salt diet (HSD) increases cortisol levels in man. We hypothesized HSD induces catabolic urea generation to facilitate renal water conservation during dietary salt excretion.

Methods:
16 male mice were pair-fed either a low-salt diet (<0.1% NaCl plus tap water) or a HSD (4% NaCl plus 0.9% saline water) for two weeks. We investigated urinary concentration, body weight, MRI lean body mass, tissue urea levels, and enzymatic urea and creatine generation in liver, skeletal muscle, skin and kidney.

Results:
HSD increased renal Na clearance, decreased urea clearance and resulted in urinary Na concentration. HSD reduced body weight and lean body mass, indicating catabolic muscle wasting. This catabolic state was paralleled by urea accumulation and increased arginase activity in liver and in skeletal muscle, while renal urea excretion was reduced. Expression of L-arginine:glycine amidinotransferase (AGAT), which generates the creatine precursor guanidino-acetate and is representative for anabolic liver/muscle metabolism, was selectively reduced in the livers of HSD mice.

Conclusion:
HSD induces urea production and body urea accumulation to allow for water-economizing urinary Na concentration. The liver regulates Na homeostasis and induces catabolism by favoring urea over creatine production. Catabolic skeletal muscle serves as a glutamine reservoir for urea generation in HSD mice, resulting in sarcopenia.

Perspectives:
Cachexia is associated with increased cardiovascular mortality and heart failure in humans. Our findings link this condition to catabolic urea osmolyte generation for maintaining Na balance.
Abstract P255: Age and Structural Alterations in Subcutaneous Small Resistance Arteries of Hypertensive Patients
Authors: De Ciuceis Carolina , Rossini Claudia , Agabiti Rosei Claudia , Porteri Enzo , Gavazzi Alice , Pileri Paola , Caletti Stefano , Agabiti Rosei Enrico , Rizzoni Damiano
Published: March 2022

Background:
It was proposed that early vascular ageing may be an important mechanism of vascular damage in large conductance arteries. However it is not known whether aging may also affect small resistance artery morphology.

Patients and Methods:
For this reason, we investigated 100 patients with essential hypertension. Secondary forms of hypertension were excluded according to standard clinical evaluations and biochemical or instrumental assessments. In all patients, an evaluation of small resistance arteries morphology was performed by a wire micromyographic approach (Mulvany’s technique). A small amount of subcutaneous tissue was obtained by local biopsy or during election surgery and subcutaneous small resistance arteries were dissected and mounted on a myograph; the media to lumen ratio (M/L) was then measured.

Results:
The age range of our populaton was 22-81 years, with a mean value of 57±12 years; 14% of them were current smokers, 32% had alterations in lipid patterns, none of them had diabetes mellitus, 58 were males and average blood pressure values were 156/95±19/12 mmHg.

We found a significant correlation between M/L and age (r=0.30, p=0.002): the statistical significance of the correlation persisted after correction for counfounding variables (gender, serum cholesterol, smoking status, serum glucose, systolic or diastolic blood pressure values). A statistically significant inverse correlation was also observed between internal diameter and age (r=-0.20, p=0.046), while the correlation beween age and media thickness did not reach statistical significance (r=0.09, p=0.37).

Conclusion:
Our data suggest that age may affect microvascular structure in hypertensive patients. It is also possible that hypertension may anticipate the effects of physiological aging, and this should be explored in a relatively large population of normotensive subjects.
Abstract P612: TLR3-Induced Placental miR-155 Contributes to Preeclampsia by Augmenting Inflammation
Authors: Bounds Kelsey R , Crutsinger Collin P , Chiasson Valorie L , Chatterjee Piyali
Published: March 2022

Hypertensive disorders of pregnancy including preeclampsia (PE) affect ~15% of all pregnancies. We have previously demonstrated that excessive maternal immune system activation via Toll-like receptor 3 (TLR3) contributes to the development of PE-like symptoms such as hypertension, endothelial dysfunction, and proteinuria in mice only when pregnant. Recently, increased miR-155 (an inflammation-related miRNA) expression was reported in placentas from women with PE compared to normotensive women suggesting a pathophysiological role of miR-155 in PE. Whether miRNAs play a role in the etiology of PE by augmenting inflammation is unknown. We confirmed miR-155 expression was up-regulated in placentas of poly I:C (a TLR3 agonist) treated wild type mice by microarray (P-PIC: 1.56 fold, p<0.5 vs controls) and qRT-PCR analyses (P-PIC: 3.15 fold, p<0.05 vs. controls). Based on our data we hypothesized that TLR3 activation induces placental miR-155 expression that augments maternal inflammation leading to PE. Transcription factors NF-κB and AP-1 which are known to bind to the promoter of miR-155 were increased in placentas of P-PIC mice. TargetScan, a target identification algorithm predicted suppressor of cytokine signaling 1 (SOCS1) as a putative target of miR-155. SOCS1 prevents overactivation of immune responses but failed to increase in P-PIC mice. ICAM, a marker of inflammation increased significantly in P-PIC mice. P-PIC TLR3 KO mice did not develop hypertension and proteinuria as well as did not exhibit increased placental levels of NF-κB, AP-1, miR-155 expression and ICAM or decreased levels of SOCS1. To determine the placental etiology, human cytotrphoblasts (CTBs) were treated with poly I:C and NF-κB, AP-1 transcription factor levels and miR-155 expression were increased significantly while SOCS1 levels decreased. Together, these data suggest that TLR3 activation increases placental miR-155 expression which downregulates SOCS1 levels and increases inflammation which may contribute to the development of PE.
Abstract P620: Renal Nerve Denervation Modulates GABA-ergic Input into Paraventricular Nucleus of the Hypothalamus and Exerts a Long Term Antihypertensive Effect in Hypertensive Mice Associated with Chronic Kidney Disease
Authors: Nishihara Masaaki , Hirooka Yoshitaka
Published: March 2022

Background:
Sympathoexcitation plays an important role in the pathogenesis of hypertension with chronic kidney disease (CKD). In hypertension, the paraventricular nucleus of the hypothalamus (PVN) in the brain controls the sympathetic outflow through GABA-ergic mechanisms. The renal nerve denervation (RDN) exerts a certain long term antihypertensive effect; however the precise mechanism is not fully elucidated. We aimed to clarify whether RDN modulates sympathetic outflow through GABA-ergic mechanisms in the PVN in hypertensive mice with CKD.

Methods and Results:
In 5/6-nephrectomized ICR-mice (Nx) at 4-weeks after nephrectomy, systolic BP (SBP) was significantly increased (vs. Sham, 143±2 vs. 109±2mmHg, n=12-24, p<0.01), accompanied by sympathoexcitation (urinary norepinephrine (uNE): vs. Sham, 424±26 vs. 226±17μg/24hrs, n=12-24, p<0.01). We performed RDN or sham operation and divided into 3 groups (Sham-sham, Nx-sham, and Nx-RDN, n=12 for each). At 2-weeks after RDN, SBP was significantly decreased (123±2 vs. 136±2 mmHg, n=12 for each, p<0.01), and urinary sodium excretions were increased (0.64±0.04 vs. 0.47±0.03 mmol/24hrs, n=8 for each, p<0.01) in Nx-RDN compared with those in Nx-sham. The uNE levels were not different between two groups. At 6-weeks after RDN, SBP was kept decreasing (125±1 vs. 140±3 mmHg, n=12 for each, p<0.01) and uNE levels were also decreased (386±32 vs. 541±35 μg/24hrs, n=12 for each, p<0.01) in Nx-RDN compared with those in Nx-sham. The urinary sodium excretions were not different between two groups. Bicuculline (GABA-A receptor antagonist, 50pmol) microinjection into PVN increased mean arterial pressure (MAP) and lumbar sympathetic nerve activity (LSNA) in all groups. The pressor responses and the change in LSNA were significantly attenuated in Nx-sham (vs. Sham-sham, [[Unable to Display Character: ∆]]MAP/baseline MAP [%], 33±4 vs. 66±6 %; [[Unable to Display Character: ∆]]LSNA (%baseline), 57±7 vs. 112±8 %, p<0.01, n=6 for each), but were enhanced in Nx-RDN at 6-weeks after RDN (vs. Nx-sham, [[Unable to Display Character: ∆]]MAP/baseline MAP [%], 60±6 vs. 33±4 %; [[Unable to Display Character: ∆]]LSNA (%baseline), 94±5 vs. 57±7 %, p<0.05, n=6 for each).

Conclusion:
These data indicate that augmented GABA-ergic input into PVN induced by RDN, at least in the late phase, is involved in antihypertensive action in hypertensive mice with CKD.
Abstract P624: Characterisation of the Chemerin Axis in the Human Vasculature
Authors: Kennedy Amanda , Kuc Rhoda , Maguire Janet , Davenport Anthony
Published: March 2022

Objective:
The peptide chemerin is an emerging novel vasoconstrictor which has been implicated in altering vascular function in animal models. CMKLR1 is a known chemerin receptor which functions predominately in immune response. Chemerin has also been proposed as the ligand for orphan receptor GPR1, which our group has recently confirmed. We tested the hypothesis that chemerin was vasoactive in human vessels and identified which receptor mediated this action.

Method and Results:
Immunohistochemistry revealed that chemerin was expressed in endothelial cells and receptors CMKLR1 and GPR1 were expressed in the smooth muscle cells of human vessels. Quantitative PCR analysis of human vessels supported this with CMKLR1 10 fold more abundant than GPR1. C-terminal fragment chemerin149-157 (C9) caused a concentration dependent contraction in isolated endothelium denuded human saphenous vein (hSV) with a pD2=7.4±0.3 (n=4), which was shifted to the right by antagonist CCX832 (100mM), pD2=6.1±0.2 (n=3). Full characterisation of the novel antagonist CCX832 with receptor-transfected cells confirmed it to be selective for the CMKLR1 receptor, and had no effect on GPR1. In β-arrestin recruitment assays, CCX832 was functionally active at the CMKLR1 receptor causing a rightward shift of the C9 response, logKD=-8.2±0.1 (n=3); however no effect was seen at the GPR1 receptor. Radiolabelled competition binding experiments showed that CCX832 competes for binding with [125I]-C9 at the CMKLR1 receptor, pIC50=9.0±0.1 (n=5), however it does not at the GPR1 receptor. In competition binding experiments on human saphenous vein homogenate, CCX832 competes for binding with [125I]-C9, pIC50=8.6±0.4 (n=6). This is in agreement with the in vitro pharmacology data, that chemerin’s vasoactivity is mediated by CMKLR1. Data is expressed as mean±SEM.

Conclusion:
These data support the emerging role of chemerin as an endogenous vasoconstrictor through its receptor CMKLR1. It identifies for the first time the components of the chemerin axis in the human vasculature, and confirms that chemerin causes vasoconstriction of human vessels. This study identifies a novel therapeutic drug target for hypertension in humans.
Abstract P626: Amine Oxidase Activity in Mesenteric Perivascular Adipose Tissue Adipocytes is Mediated by Semicarbazide Sensitive Amine Oxidase
Authors: Ayala-Lopez Nadia , Thompson Janice M , Watts Stephanie W
Published: March 2022

Perivascular adipose tissue (PVAT) is important in regulating vascular tone and reduces contraction of vessels to various agonists. PVAT is composed of adipocytes and the stromal vascular fraction (SVF), which contains immune cells, neurons, endothelial cells, fibroblasts and preadipocytes. Adipocytes, the main cell type in PVAT, contain various amine oxidases. We hypothesized that amine oxidases in mesenteric PVAT would metabolize vasoactive amines. To determine the primary enzyme(s) responsible for the amine oxidase activity in PVAT, an Amplex Red assay was performed. We added tyramine, a substrate for monoamine oxidase-A & B (MAO-A, MAO-B) and semicarbazide sensitive amine oxidase (SSAO), to cell and tissue fractions from male Sprague Dawley rats to test for H
2
O
2
production as an indicator of oxidase activity. Oxidase activity in mesenteric PVAT increased from 7.92±2.27 (vehicle) to 20.83±5.30 with the addition of tyramine (p<0.05) and from 9.30±2.55 to 24.60±6.38 in adipocytes (p<0.05; pmol/min·mg±SEM; N=5). In both the mesenteric PVAT and isolated adipocytes, the oxidase activity was reduced to baseline levels (vehicle) by pre-incubating with the SSAO inhibitor semicarbazide (1 mM; PVAT: 8.49±2.56, adipocytes: 9.43±3.21; p<0.05; N=4-5). No reduction in oxidase activity in both the mesenteric PVAT and the isolated adipocytes was observed when the MAO-A inhibitor clorgyline or the MAO-B inhibitor pargyline (1 μM; a concentration at which each drug is specific for their respective enzyme) were used. Westerns revealed that mesenteric PVAT adipocytes had the highest expression of monomeric SSAO (~80 kDa; 124.75±30.71%) compared to the whole PVAT (66.15±9.92%), blood vessels (48.2±6.89%) and the SVF (38.76±11.65%) as quantified by percent density relative to β-actin using protein isolated from male Sprague Dawley rats (p<0.05; values given as %β-actin±SEM, N=4). Oxidase activity within the SVF remains to be determined. These findings support that most of the oxidase activity in mesenteric PVAT can be attributed to SSAO and the adipocyte is an abundant source of SSAO. Due to the proximity of PVAT to the blood vessel, oxidase activity in PVAT through SSAO may alter vascular tone by metabolizing vasoactive amines and through the production of H
2
O
2
.
Abstract P642: Impact of Urinary Endothelin-1 on Derangements in Stress-Induced Pressure Natriuresis
Authors: Mathur Shreya , Pollock David M , Pollock Jennifer S , Harshfield Gregory A
Published: March 2022

Sodium retention during stress (retainer) is known to increase the risk of hypertension and other diseases. Our group has previously shown that angiotensin II type I receptor blockade improves natriuresis in retainers. Since our pre-clinical studies demonstrate that angiotensin II inhibits endothelin-1 (ET-1) dependent natriuresis, we predict that ET-1 may be linked to the response. We hypothesize that reduction in urinary (renal) ET-1 accounts for derangements in sodium handling under stress, a link never before explored in a large human cohort. We evaluated urinary ET-1 and albumin excretion in 4 studies of stress-induced pressure natriuresis, of which 3 were observational studies with 776 healthy youth (15 - 19 years) enrolled in a 5 hr protocol (1 hr of mental stress before and after 2 hrs of rest). The 4th study involved 213 African American adults (18 - 54 years) in a double blind crossover trial comparing irbesartan (angiotensin II type I receptor antagonist) to placebo. The protocol entailed 7 days of vehicle (placebo or irbesartan 150 mg P.O.) followed by a 3 hr protocol (1 hr of rest before and after 1 hr of mental stress). In all studies, 60 min urine samples were obtained. Subjects were grouped as retainers or excreters if they retained or excreted sodium under stress. In the observational studies, mean change in ET-1 between stress and baseline was significant (p<0.001), being negative (mean= -0.0154 pmol/min) in retainers but positive (mean= 0.0194 pmol/min) in excreters. ET-1 excretion was significantly higher (p<0.028) in retainers than excreters at baseline but significantly lower in retainers under stress (p<0.0001). ET-1 excretion continued to decline in retainers during recovery but returned to pre-stress levels in excreters. Albumin excretion and albumin to creatinine ratio were significantly higher in retainers (p<0.046, p<0.008, respectively). During stress, the irbesartan group had significantly higher ET-1 excretion than placebo (p<0.001). Thus loss of ET-1-dependent natriuresis may account for sodium retention during stress and correction of sodium handling re-establishes ET-1-mediated natriuresis. Retainers have both lower ET-1 excretion and increased albuminuria, suggesting renal impairment and risk for future diseases.
Abstract 008: Leptin Treatment Attenuates Vascular Dysfunction and Inflammation in Mouse Model(s) of Acquired Lipodystrophy via Reducing Nox1-derived Ros
Authors: Bruder-Nascimento Thiago , Chen Weiqin , Stepp David , Belin de Chantemèle Eric J
Published: March 2022

Although extremely efficient at suppressing HIV replication, highly active antiretroviral therapy (HAART) induces lipodystrophy, a metabolic disorder characterized by an abnormal adipose tissue distribution, reduced leptin levels and vascular dysfunction. Leptin replacement therapy (LRT) is currently used to improve metabolic function in patients suffering from congenital lipodystrophy. Here, we analyzed whether LRT restores vascular function and inflammation in mice treated with the antiretroviral agent, ritonavir (Rit). Four weeks of Rit reduced body weight [control (C): 28.4±0.5 vs. Rit: 24.4±0.2g*, *P<0.05] and fat mass (C: 10±1 vs. Rit: 6.5±1 %*) confirming that it induces lipodystrophy. Rit impaired aortic endothelial function [Relaxation to acetylcholine: C: 74±4 vs. Rit: 21±15%*), increased ROS producing enzymes (NOX1 and NOXA1), induced vascular inflammation (increased IL-1β, MCP-1, GATA Binding Protein 3 and INF-γ gene expression) and increased TBARS levels. ROS scavenging via tempol or GKT137117, (Nox1/4 inhibitor) pre-incubation blunted endothelial dysfunction. LRT (10μg/day/7 days, osmotic mini-pump), at the end of the 3-week Rit, restored endothelial function, reduced Nox1 and NOXA1 gene expression and vascular inflammation. NOX1 deficiency in Nox1 KO mice protected mice from Rit-induced endothelial dysfunction and vascular inflammation. Increasing endothelial leptin sensitivity via specific deletion of protein tyrosine phosphatase 1B (Ptp1b) in endothelial cells (
Ptp1b-/-EC
mice) protected mice from Rit-induced endothelial dysfunction and reduced Nox1 and NOXA1 gene expression, and vascular inflammation. To address the relevance of these observations to other forms of acquired lipodystrophy, experiments were repeated in mice in which lipodystrophy was induced at 8 week of age, by the deletion of Bscl2, a gene involved in adipocyte maturation. Bscl2 deletion reduced fat mass, and induced endothelial dysfunction via ROS-mediated mechanisms. Again, LRT reverted endothelial dysfunction by downregulating Nox1 expression. All together, these data presents leptin as a key regulator of endothelial oxidative stress level and as a potential avenue for the treatment vascular disease.
Abstract 013: The Molecular Program of Renin
Null
Cells
Authors: Brown Robin Isadora , Oka Masafumi , Martinez Maria F , Medrano Silvia , Gomez R A
Published: March 2022

Mice homozygous for the
Ren1c
gene disruption (

Ren1c
-/-

) display structural and functional defects characterized by a poorly developed renal medulla, urine concentration failure, polydipsia, polyuria, hydronephrosis, renal failure and anemia. Underlying this complex phenotype, mice exhibit unique renal vascular abnormalities, including concentric arteriolar hypertrophy. Using lineage and promoter activity tracking, we showed that renin
null
cells contribute directly to blood vessel thickening and distribute throughout renal arterial trees. We hypothesize that renin
null
cells synthesize factors that lead to arterial thickening and maintain an active molecular memory of the renin phenotype.

To test this hypothesis, we performed RNA-seq of YFP sorted single cells from kidneys of

Ren1c
-/-
; Ren1c

-YFP (KO) and

Ren1c
+/+
; Ren1c

-YFP (wildtype, WT) adult mice. We captured individual cells using a microfluidic C1 system and sequenced cDNA from cellular mRNA. We also performed ATAC-seq of KO cells to identify open chromatin regions available for transcription factor binding.

KO and WT cell populations were distinct, with the average Euclidean distance between genotypes 1.9x greater than within genotypes. Differential expression analysis revealed that KO cells upregulated 1395 genes and downregulated 364 compared to WT (log fold change > 1, p < 0.05). Among the upregulated genes were 107 potentially secreted proteins and 64 putative transcription factors. Secreted protein genes were enriched for GO terms such as angiogenesis and cell proliferation (p < 0.01), suggesting a possible cause of arteriolar abnormalities in KO kidneys. We identified several upregulated transcription factors, including Foxp1, Stat1, and KLF family genes, that had predicted binding motifs in open chromatin regions, such as upstream the Ren1 gene (p < 1.0E-10). These factors are key candidates for regulating the molecular memory of the renin cell.

This study shows that over activation of the renin program due to lack of renin causes expression of a distinct suite of genes that may be responsible for vascular pathologies observed in KO mice. These data also provide insight into how the cell regulates the renin cell program in response to chronic stimuli that jeopardize homeostasis.
Abstract 015:
Ctcf
is Required for Renin Expression and Maintenance of the Structural Integrity of the Kidney
Authors: Martinez Maria F , Li Minghong , Medrano Silvia , Gomez R A
Published: March 2022

Renin is crucial for the regulation of blood pressure and fluid electrolyte homeostasis. The transcriptional machinery that regulates renin expression and thus determines the identity of renin cells is not completely understood. CCCTC-binding factor (
Ctcf
), is an important chromatin organizer of genes that confer cell identity and tissue-specificity. Because
Ctcf
binds to several sites in the neighborhood of the renin locus, we hypothesized that
Ctcf
may regulate renin expression. To test this hypothesis, we generated mice with conditional deletion of
Ctcf
in cells of the renin lineage (
Ctcf
cKO).
Ctcf
cKO mice showed fewer renin-positive cells as shown by immunostaining, and a 70% reduction of renin mRNA levels when compared to control mice (0.292 ± 0.246
vs
1.003 ± 0.097,
p<0.001
). In addition, plasma renin levels were significantly decreased in
Ctcf
cKO versus control mice (15276.544 ± 6778.735 pg/ml
vs
62321.62 ± 21881.99 pg/ml,
p<0.001
). Consistent with reduced renin levels,
Ctcf
cKO mice had lower mean arterial pressures (60.09 ± 3.12 mmHg
vs
75.07 ± 3.06 mmHg,
p<0.001
). The kidney/body weight ratio in
Ctcf
cKO mice was markedly reduced (1.05 ± 0.228 %
vs
1.29 ± 0.074 %,
p<0.05
), indicating a more pronounced effect in kidney than in somatic growth (20.09 ± 1.47 g
vs
22.95 ± 2.95 g,
p<0.05
). Masson’s trichrome staining revealed interstitial fibrosis coinciding with cortical depressions in
Ctcf
cKO kidneys. Moreover, PAS staining of
Ctcf
cKO kidneys showed dilated tubules with intraluminal casts, and areas with crowded sclerotic and crescent glomeruli surrounded by disorganized packed cells. Finally,
Ctcf
cKO mice exhibited renal failure evidenced by increased BUN (44.29 ± 17.62 mg/dL
vs
26 ± 3.39 mg/dL,
p<0.05)
, and inability to concentrate their urine (448 ± 85.57 mOsm/kg
vs
1519.33 ± 382.39 mOsm/kg,
p<0.001
). In summary, deletion of
Ctcf
in cells from the renin lineage leads to decreased endowment of renin-expressing cells accompanied by decreased circulating renin, hypotension, severe morphological abnormalities of the kidney and ultimately renal failure. We conclude that
Ctcf
is necessary for the appropriate expression of renin, control of renin cell number and structural integrity of the kidney.
Abstract 017: Mitoprotection Attenuates Myocardial Vascular Impairment and Decreases Coronary Retention of Endothelial Progenitor Cells in Swine Metabolic Syndrome
Authors: Yuan Fang , Hedayat Ahmad F , Ferguson Christopher M , Tang Hui , Lerman Amir , Lerman Lilach O , Eirin Alfonso
Published: March 2022

Introduction:
The metabolic syndrome (MetS) leads to cardiac vascular injury, which may reflect in increased retention of endothelial progenitor cells (EPC). Coronary endothelial cell (EC) mitochondria partly regulate vascular function and structure. We hypothesized that chronic mitoprotection would preserve EC mitochondria and attenuate coronary vascular injury and dysfunction in swine MetS.

Methods:
Pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with the mitochondria-targeted peptide elamipretide (ELAM, 0.1mg/kg SC q.d), and lean controls (n=6 each). Coronary artery and sinus blood samples were collected. EC mitochondrial density (electron microscopy), endothelial nitric oxide (eNOS) immunoreactivity (staining), myocardial microvascular density (3D micro-CT), and coronary endothelial function (organ bath) were assessed ex-vivo. The number and arteriovenous gradient of CD34+/KDR+ EPC was calculated by FACS (a negative gradient indicating EPC retention).

Results:
MetS and MetS+ELAM pigs developed similar MetS (obesity, hyperlipidemia, insulin resistance, and hypertension). EC mitochondrial density decreased in MetS compared to lean, but normalized in MetS+ELAM. ELAM also improved eNOS immunoreactivity, subendocardial microvascular density, and coronary endothelial function. ELAM-induced vasculoprotection was reflected in decreased coronary retention of EPCs.

Conclusions:
These observations underscore the role of mitochondria in regulating the cardiac circulation in experimental MetS, and the benefits of mitoprotection to preserve EC myocardial microvascular integrity.
Abstract 026: Early-onset Preeclampsia is Associated With Left Ventricular Concentric Remodeling at 1-month Post-partum Follow-up
Authors: Colussi GianLuca , Catena Cristiana , Driul Lorenza , Pezzutto Francesca , Fagotto Valentina , Sechi Leonardo A
Published: March 2022

Preeclampsia (PE) is associated with persistent cardiac abnormalities and a high cardiovascular risk during the woman life. Early onset PE presenting before the 34
th
gestational week is a more severe form of disease, but its relevance on cardiac abnormalities is unknown. The aim of this study was to assess cardiac structure and function in women with PE without preexistent hypertension. One month after delivery, the clinical and echocardiographic variables of 65 preeclamptic women (age 36±6 y), 37% of which with early PE, was compared with a group of 30 normotensive (age 37±6 y), 16 hypertensive (age 40±5 y) nonpregnant women, and 6 women with normal pregnancy (age 36±5 y) who were matched for age and height. Despite comparable antihypertensive treatment, women with early PE had lower 24-hour average systolic and diastolic blood pressure than those with late PE (125±11 Vs 136±15). Left ventricle (LV) relative wall thickness was significantly greater in women with early PE (0.36±0.06) than in those with late PE (0.31±0.07; p<0.05) and normotensive (0.30±0.06; p<0.05) women, but not hypertensive women (figure). Women with early and late PE had comparably greater LV mass and worse diastolic function as assessed by the E/A ratio (PE 1.6±0.4 Vs 1.8±0.4 P<0.01) and isovolumic relaxation time (PE 96±28 Vs 73±12 msec, P<0.001) than normotensive women. In conclusion, women with early onset PE have more pronounced LV concentric remodeling than women with late onset PE. This observation could account for the greater cardiovascular risk of these patients and might prompt the use of antihypertensive drugs specifically acting on LV remodeling.
Abstract 029: Nox5 is a Pro-contractile Nox Isoform - Implications in Vascular Contraction and Cardiac Fibrosis
Authors: Montezano Augusto C , Camargo Livia L , Harvey Adam P , Rios Francisco J , Holterman Chet E , Kennedy Christopher R , Touyz Rhian M
Published: March 2022

The functional significance of Nox5 is unknown. Considering the fact that Nox5 is closely associated with changes in [Ca
2+
] and that it generates ROS, both of which are important in contraction, we questioned whether Nox5 plays a role in pro-contractile signaling and whether it influences vascular function. We generated humanised Nox5 mice with Nox5 expressed in a VSMC-specific manner (Nox5+SM22+). Vascular contraction was measured by myography. ROS production was assessed by HPLC, amplex red and ELISA. Protein levels were evaluated by immunoblotting. Fibrosis was assessed by Picro Sirius red staining and polarized microscopy. Contraction to U46619 was increased in Nox5+/SM22+ mice (5.8±0.3 mN vs WT: 4.2±0.2 mN, p<0.05), an effect blocked by a NAC (ROS scavenger), calmidazolium (calmodulin inhibitor), dantrolene (ryanodine receptor Ca
2+
channel inhibitors) and CDN1163 (SERCA channel activator). ONOO
-
levels were increased in vessels from Nox5+/SM22+ (5.8±0.9 vs WT 3.4±0.1 AU/mg, p<0.05). ZIPK is an important regulator of MYPT1 inactivation. In vessels from Nox5+/SM22+ mice, ZIPK activation was increased (58.6±3.64 vs 27.73±7.64 AU, p<0.05). VSMC-Nox5 exhibited increased cardiac levels of superoxide (WT: 606.3±78.5 vs 1456.0±184.8 nmol/mg of protein), H2O2 (WT: 11.1±1.3 vs 23.88±5.1 μM/μg of protein) lipid peroxidation (WT: 0.70±0.09 vs 1.18±0.18 nmol/ μg of protein), cardiac fibrosis (WT: 3.46±1.71 vs 4.39±0.04 AU), p38 MAPK activation (WT:0.98±0.04 vs 1.61±0.12 AU) and fibronectin expression (WT:1.23±0.07 vs 2.31±0.29 AU) (p<0.05). Moreover, peroxiredoxin oxidation was increased (WT: 1.43±0.4 vs 6.28±2.0 AU, p<0.05). In VSMCs, downregulation of Nox5, but not Nox1,2,4, by siRNA was associated with reduced phosphorylation of MLC20 and MYPT1. In conclusion, our results demonstrate that Nox5 regulates vascular contraction through processes that involve , ROS, calmodulin, ryanodine and ER-Ca
2+
channels. Nox5 may be an important regulator of the contractile machinery in VSMCs. In addition, VSMC-Nox5 induces oxidative stress in the heart, leading to fibrosis. Our study defines a novel role for Nox5 as a pro-contractile Nox isoform that may have important implications in conditions associated with vascular hypercontractility.
Abstract 036: Regulation of sFlt-1 Splicing by U2AF and JMJD
Authors: Eddy Adrian C , Chapman Heather , George Eric M
Published: March 2022

Preeclampsia (PE) is a gestational disorder defined by new-onset hypertension and maternal endothelial dysfunction. Though its origins are unclear, placental ischemia is thought to be the central cause. In response, the placenta releases pathogenic factors, such as the anti-angiogenic soluble form of the VEGF receptor Flt-1 (sFlt-1), into the maternal circulation. sFlt-1 is a soluble splice variant of the VEGF receptor Flt-1, acting as a VEGF sink in the circulation. The regulation of sFlt-1 splicing is not completely understood, but it is proposed that the splicing factor U2AF65 promotes splicing, while JMJD6 opposes it by degrading U2AF65. Here, we test the hypothesis that decreased expression of U2AF65 will decrease sFlt-1 release, while decreased JMJD6 will increase sFlt-1 release. Using immortalized placental trophoblasts (BeWo), siRNA was used to knock down the expression of U2AF65, confirmed by RT-qPCR (1.014
+
0.12 vs 0.379
+
0.131; p<0.05). After transfection, cells were incubated at 8% and 1% oxygen for 24 hours, mimicking the conditions found in the healthy and ischemic placenta respectively. As hypothesized, U2AF65 knockdown caused a 52% decrease in released sFlt-1 compared to controls in hypoxia (56.8
+
9.4 pg/mL vs 118.9
+
9.2 pg/mL; p<0.05). siRNA successfully reduced JMDJ6 mRNA (1.008
+
0.09 vs 0.3698
+
0.04; p<0.05) and demonstrated undetectable protein levels. Unexpectedly, this resulted in a 33.8% decrease (342.7
+
35.8 pg/mL vs 517.8
+
15.4 pg/mL; p<0.005) in the amount of sFlt-1 release from cells in hypoxia and a 49% decrease (246.2
+
23.6 pg/mL vs 482.8
+
39.4 pg/mL; p<0.001) in normoxia compared to their respective controls. We also measured both U2AF65 and JMJD6 in the placentas of control rats and those that had chronic placental ischemia due to the Reduced Uterine Perfusion Pressure (RUPP) procedure. RUPP placentas showed a 61.6% increase in U2AF65 compared to controls (161.6
+
11.6 AU vs 100
+
9.8 AU; p<0.005) and a 121% increase in JMJD6 in RUPP placentas (221.1
+
30.4 AU vs 100
+
40.9 AU; p= 0.0549). Based on these data, we speculate that both JMJD6 and U2AF65 are crucial for the production of sFlt-1 by affecting alternative splicing. Targeting these factors could prove a useful approach for reducing sFlt-1 production in the preeclampsia patient.
Abstract 038: MiR-210 Overexpression Induces sFlt1 Production and Contributes to Preeclampsia-like Symptoms in Pregnant Mice
Authors: Chiasson Valorie L , Bounds Kelsey R , Pan Lu J , Engelbrecht Rachel M , Chatterjee Piyali
Published: March 2022

Preeclampsia (PE) is an obstetric complication that is diagnosed by hypertension and proteinuria at or after 20 weeks of gestation. PE is a multifactorial disease, but it is widely accepted that impaired proangiogenic factors such as vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and antiangiogenic factor such as soluble FMS-like tyrosine kinase-1 (sFlt1) or VEGF receptor-1 balance during pregnancy is linked to PE. sFlt1, a splice variant of Flt1 binds to either VEGF or PlGF and blocks their action. In addition, placental angiogenesis is known to be regulated by specific miRNAs that are typically dysregulated during PE highlighting the importance of these miRNAs in pregnancy disorders. miR-210 is upregulated in placentas and serum/plasma of PE women and is also known to modulate angiogenic pathways. Therefore, we hypothesized that miR-210 overexpression contributes to the development of PE-like symptoms by altering the angiogenic/antiangiogenic pathway. To this end, we demonstrate increased systolic blood pressure (SBP: miR-210 TG P = 115 ± 2 mm Hg vs. WT P = 95 ± 2 mm Hg, p<0.05), endothelial dysfunction and proteinuria in pregnant miR-210 TG mice as compared to control WT mice. Placental sFlt-1 levels increased and PlGF levels decreased in miR-210 TG mice as determined by immunoblotting. Immunohistochemistry (IHC) analysis demonstrated an increase in sFlt1 and a decrease in PlGF immunoreactivity in miR-210 TG placentas. Pregnant miR-210 TG mice exhibited increased serum levels of sFlt-1 as well as decreased PlGF as compared to pregnant WT mice. Furthermore, immunohistological analysis of stained human placental sections revealed an increase in sFlt-1 and a decrease in PlGF in pregnant miR-210 TG as compared to pregnant WT mice. To determine the placental etiology, human cytotrophoblasts (CTBs) overexpressing miR-210 demonstrated a decrease in PlGF while inhibition of miR-210 in CTBs using anti-miR-210 oligos increased the PlGF expression as determined by immunoblotting. These data taken together suggest that miR-210 modulates the angiogenic/antiangiogenic pathway and contributes to PE-like features in mice. Thus, targeting miR-210 by delivering anti-miR-210 oligos may prevent PE-like symptoms in pregnant women.
Abstract 046: Chronic Estrogen Replacement Prevents the Increase in Blood Pressure in Female IUGR Offspring at 12 Months of Age
Authors: Davis Gwendolyn K , Newsome Ashley D , Ojeda Norma B , Alexander Barbara T
Published: March 2022

Low birth weight
(LBW
) is associated with an earlier age at menopause and a greater prevalence of hypertension in women in later life. Using a rodent model of LBW induced by placental insufficiency, previous studies from our lab indicate that intrauterine growth restriction (
IUGR
) programs a significant increase in blood pressure (
BP
) by 12 months of age in female IUGR offspring. Female IUGR are also acyclic by 12 months of age, or 6 months prior to control indicative of early reproductive senescence. The mechanisms involved in menopause related cardiovascular risk are not clear. Furthermore, the link between birth weight and the greater prevalence for hypertension in LBW women in later life is not known. Female IUGR exhibit a shift in the testosterone to estradiol ratio at 12 months of age. Thus, this study tested the hypothesis that a loss of estradiol (
E2
) contributes to the increase in BP in female IUGR offspring that develops by 12 months of age. Female rats received vehicle or 17β-Estradiol valerate minipellets (1.5mg for 60-day release) for 6 weeks starting at 12 months of age with BP measured in conscious, chronically catheterized rats following treatment (2way ANOVA; Tukey’s multiple comparison). BP was significantly increased in vehicle-treated IUGR relative to vehicle-treated Control (*, p<0.05); an increase that was abolished by chronic E2 in IUGR (**, p<0.05). Uterine weight, a crude marker of E2, was significantly increased by chronic E2 in both Control and IUGR E2-treated offspring (**, p<0.05). Therefore, this study indicates that E2 withdrawal after early reproductive senescence contributes to the increase in BP that develops in later life in female IUGR offspring.