bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 229: A Critical Role of Group IV Cytosolic Phospholipase A2α in the Development of Ang II-induced Hypertension and Associated Cardiovascular Pathophysiology
Authors: Khan Nayaab S , Jennings Brett L , Song Chi Young , Estes Anne M , Bonventre Joseph V , Malik Kafait U
Published: March 2022

This study was conducted to determine the contribution of Group IV cytosolic phospholipase A2α (cPLA2α) in the development of angiotensin (Ang) II-induced hypertension and associated pathophysiology. Eight week old male wild type (cPLA2α
+/+
) and cPLA2α knockout (cPLA2α
-/-
) mice were infused with Ang II (750 ng/kg/min) or its vehicle for 2 weeks and systolic blood pressure (SBP) was measured weekly by the tail cuff method. Ang II increased SBP in cPLA2α
+/+
mice to a greater degree than in cPLA2α
-/-
mice (125 ± 2 to 186 ± 7 mmHg vs. 125 ± 2 to 132 ± 2 mmHg respectively, P < 0.05). The increase in SBP in Ang II infused cPLA2α
+/+
mice was associated with cardiac hypertrophy, measured by heart to body weight ratio (5.0 ± 0.3 vehicle vs. 7.1 ± 0.4 Ang II, P < 0.05), which was reduced by 26.0 ± 3.9 % (P < 0.05) in cPLA2α
-/-
mice. Ang II caused cardiac fibrosis, as indicated by accumulation of intracardiac α-smooth muscle actin- and transforming growth factor-β-positive cells, and collagen deposition in cPLA2α
+/+
but not cPLA2α
-/-
mice. Ang II infusion produced a maximal increase in the response of aortic rings to phenylephrine (2.5 ± 0.5 mN vehicle vs. 6.0 ± 0.7 mN Ang II, P < 0.05) and endothelin-1 (5.1 ± 1.0 mN vehicle vs. 9.5 ± 1.0 Ang II, P < 0.05), and caused endothelial dysfunction in cPLA2α
+/+
mice as indicated by a decrease in maximal relaxation in response to acetylcholine (92.0 ± 2.3 % vehicle vs. 55.1 ± 5.7 % Ang II, P < 0.05), measured by wire myograph. The vascular responses to PE and ET-1 were attenuated by 21.5 ± 8.5 % and 22.9 ± 11.4 %, respectively, and the relaxation to ACh increased by 36.9 ± 7.7 % (P < 0.05) in Ang II treated cPLA2α
-/-
mice. Aortic production of reactive oxygen species (ROS), measured by 2-hydroxyethidium fluorescence, was increased in Ang II treated cPLA2α
+/+
mice (25.3 ± 1.6 AU vehicle vs. 49.5 ± 3.9 AU Ang II, P < 0.05) but not cPLA2α
-/-
mice (22.7 ± 1.1 AU vehicle vs. 23.5 ± 1.2 AU Ang II, P < 0.05). These data suggest that cPLA2α contributes to Ang II-induced hypertension and associated cardiovascular pathophysiology, most likely due to release of arachidonic acid and activation of NADPH oxidase and generation of ROS. Thus, cPLA2α could serve as a potential therapeutic target in the treatment of hypertension and cardiac damage and vascular dysfunction associated with hypertension.
Abstract 281: Measurement of Peripheral Blood 18-oxo-cortisol Can Differentiate the Patients With Aldosterone Producing Adenoma From Those With Essential Hypertension. -omitting many steps of diagnostic workup-
Authors: Satoh Fumitoshi , Morimoto Ryo , Kudo Masataka , Iwakura Yoshitsugu , Ono Yoshikiyo , Sasano Hironobu , Nakamura Yasuhiro , Ito Sadayoshi
Published: March 2022

Backgrounds:
Primary aldosteronism (PA) is diagnosed and treated by the long steps, such as screening, confirmation testing and subtype diagnosis (computed tomography (CT) scan and adrenal venous sampling (AVS)).

Objective:
The aim of the study was to determine the role of peripheral plasma levels of 18-oxo-cortisol (p18oxoF) in discriminating aldosterone producing adenoma (APA), which is a major surgically curable subtype of PA, from essential hypertension (EH) which should be medically treated.

Patients:
The study included 79 EH patients with the aldosterone/renin activity ratio (ARR) of 9.0±0.5 (ng/dl per ng/ml/h) and 87 patients with CT-positive macro APA (ARR: 363±38.7).

METHODS:
All the unilateral APA patients diagnosed by AVS were surgically proven and histopathologically confirmed including immunohistochemical analysis of steroidogenic enzymes. We measured p18oxoF of all patients by high sensitive LC ms/ms.

Results:
APA patients showed significantly higher levels of p18oxoF and compared to EH patients. Especially, the ROC analysis of 18oxoF in CT-positive macro APA versus EH demonstrated clinically significant discrimination with higher sensitivity of 0.81 and higher specificity of 0.97 by cutoff value of 4.6 ng/dl.

Conclusions:
Peripheral plasma levels of 18-oxo-cortisol can practically differentiate the patients with APA from those with essential hypertension omitting many steps of diagnostic workup for PA.
Abstract 289: Continuum of Care of Prehospital to Hospital Hypothermia Therapy, Outcomes in a Large Prehospital System in Conjunction With a Major Metropolitan Hospital.
Authors: El Sanadi Nabil , Mansour Jason , McCormack Violet , Grubb Lynn , Brattan Paul , Heiser John , Heiser Tim , Leduc Todd , Erdman David , Engebretsen Labbe Trine , Thornton Gabriel
Published: March 2022

Introduction:
Hypothermia in patients after Return of Spontaneous Circulation (R.O.S.C.) was recommended in the 2005 American College of Cardiology/American Heart Association Guidelines. Prehospital use of cold saline (32-34°c) and ice packs to the proximal extremities, has been used for patients with R.O.S.C.. In hospital use of intravascular cooling and heart catheterization yield the best neurological and cardiac outcomes as recommended by the A.C.C./A.H.A. 2008 guidelines.

METHODS:
A retrospective chart review was conducted spanning 3/2009 to 9/2010. Including prehospital run sheets and hospital records. Prehospital provider protocols for 968 paramedics (11 municipalities) were modified to allow the providers to place ice packs at the proximal extremities and to start iced saline I.V. (temp 32-34°c) for victims of cardiorespiratory arrest with R.O.S.C. On arrival to the hospital the cardiologist and the emergency physicians started internal cooling of these patients. The cardiologist and the catheterization team diagnosed and treated preexisting coronary disease in the catheterization laboratory. Cooling was continued for twenty four hours. The patients were then evaluated to determine their level of neurological function prior to discharge.

Results:
31 patients were brought in by paramedics after starting the prehospital hypothermia protocol, after R.O.S.C. 26 men and 5 women. All patients were admitted through the emergency department to the heart catheterization laboratory where central cooling was started and coronary angiography was performed, with angioplasty and stenting was done when indicated. Cooling was at 32°c and was maintained for 24 hours. 13/31 patients expired prior to discharge (42%) from the hospital. 5/31 patients were transferred for hospice care or to long term-care facilities (16%). 12/31 patients left the hospital neurologically intact (39%).

Conclusion:
The sequential use of prehospital cooling and inhospital intravascular cooling for victims of cardiorespiratory arrest with R.O.S.C. is feasible, safe and effective. Our results for neurologically intact hospital discharges of 39%, is higher than that reported by prior investigators.
Abstract 290: Long Term Safety and Efficacy of Renal Nerve Ablation in Swine with the First Prototype of the Boston Scientific Renal Ablation System
Authors: Winsor-Hines Dawn , Tunstall Radhika , Kocur Gordon , Harrison Kent , Scherer Steve , Vrba Tony , Huibregtse Barbara
Published: March 2022

Introduction:
Early splanchnicectomy and radical sympathectomy research demonstrated that reducing sympathetic nerve activity achieved blood pressure reductions and improved long-term outcomes in hypertensive patients. These methods were abandoned due to high perioperative morbidity and mortality. A minimally invasive approach to inhibition of renal sympathetic nerves represents an attractive treatment of essential hypertension.

Methods:
Swine were evaluated at 7, 30, 60, 90 or 180 days post treatment. At each time point, 4 animals treated with bilateral renal artery radiofrequency (RF) ablation and 3 sham treated animals were assessed. A single point ablation catheter was positioned within each renal artery of anesthetized animals and 4-6 ablations/artery were delivered in a spiral pattern. At baseline and termination, urinalysis, blood chemistry and QVA was performed. Right and left kidneys and renal arteries were harvested from all animals. Renal arteries were processed for histological evaluation. Kidneys were weighed, frozen, and used for the analysis of norepinephrine levels.

Results:
No injuries were seen in tissues surrounding the renal arteries and no safety concerns were identified in the histological evaluation of the renal arteries. RF treated renal arteries showed segmental, transmural regions of minimal vessel wall injury with SMC loss. These regions showed progression of healing and complete recellularization by 60 days. All vessels had complete endothelial cell coverage at all timepoints. Minimal intimal hyperplasia was seen. There were no instances of EEL injury. Mural and adventitial inflammation were seen at 30 days, but both resolved by 60 days. No abnormalities in renal function were identified by urinalysis or serum chemistry. Efficacy of nerve ablation was determined by renal norepinephrine levels (pg/mg) and a significant decrease was seen in ablation groups relative to sham groups at every timepoint (7 days: 69% p< 0.0001, 30 days: 46% p =0.044; 60 days: 59% p<0.004; 90 days: 53% p< 0.0001; 180 days: 56% p<0.001).

Conclusions:
Renal ablation with the BSC catheter in a healthy swine model demonstrated safety and efficacy as assessed by renal artery histology and renal norepinephrine levels out to 6 months.
Abstract 3: Angiotensin II Affects Hematopoietic Stem/progenitor Cell Engraftment and Mobilization
Authors: Kim Seungbum , Scott Edward , Marulanda Jessica , Raizada Mohan
Published: March 2022

Evidence indicates that Angiotensin II (Ang II) induces proliferation of the hematopoietic stem/progenitor cells (HSPCs). However, the mechanism of its action in vivo at the level of bone marrow (BM) remains poorly understood. In the present study, we have used an innovative in vivo imaging technique to visualize mouse tibia bone so that we can elucidate the effect of Ang II on HSPC in the BM. Sca-1+, c-Kit+, Lin- (SKL) HSPCs from GFP mice were isolated and 6-10X10^3 SKL cells were injected into the lethally irradiated C57BL6 mice. SKL cells engrafted in the tibia bone were visualized in real time with the use of tibia window technique. The effect of Ang II on GFP+ SKL cells was studied by chronic infusion of Ang II osmotic minipumps (1000ng/kg/min). We observed a significant delay in the homing of GFP+ SKL cells by Ang II infusion for 7 days. In addition, the SKL cells failed to efficiently engraft to the osteoblastic niche. Consistent with this observation, colony formation unit-Spleen (CFU-S) analysis showed that extramedullary hematopoiesis was drastically reduced in Ang II treated mice. Furthermore, CX3CR1+/Gr-1+ inflammatory monocytes in the peripheral blood were increased by 47% following 2 weeks of Ang II infusion. This was associated with the significant increase of Sca-1+ CX3CR1+ cells in the BM indicating that Ang II also affects monocyte progenitor cells in the BM. Finally, we observed an increased number of BM derived monocytes in the paraventricular nucleus (PVN) of the hypothalamus in Ang II treated mice. These data demonstrate that chronic Ang II infusion inhibits homing of HSPC, prevents their engraftment into the functional BM niche and increases mobilization of monocyte progenitors into the PVN. They suggest that monocyte progenitors derived from the BM may contribute to an increase in the number of activated microglia in the PVN of hypertensive animals.
Abstract 333: Effect of Valsartan or Ramipril Addition to Amlodipine/Hydrochlorotiazide Combination on Left Ventricular Hypertrophy in Hypertensive Patients With Type 2 Diabetes
Authors: Fogari Roberto , Mugellini Amedeo , Zoppi Annalisa , Derosa Giuseppe
Published: March 2022

Objective:
to compare the effect of valsartan (Val) or ramipril (Ram) addition to amlodipine (Aml)/hydrochlorotiazide(HCTZ) combination on left ventricular (LV) mass in type 2 diabetic hypertensive patients with LV hypertrophy.

Design and Method:
a total of 180 mild to moderate hypertensive outpatients with well controlled type 2 diabetes and LV hypertrophy after a 2 week placebo period were treated with Aml 10 mg/HCTZ 12.5 mg for 4 weeks: the 154 patients whose blood pressure BP was not normalized by the dual combination (SBP > 130 mmHg and/or DBP > 80 mmHg) were enrolled in the study and randomized to the addition of valsartan 320 mg or ramipril 10 mg for 12 months. At the third month the non responder patients were discontinued. A total of 142 patients completed the study. Echocardiographic evaluation was performed at the end of the placebo period, of the amlodipine/HCTZ period and after 12 months of triple combination treatment.

Results:
systolic and diastolic BP were similarly and significantly reduced in both treatment groups (-9.2 ± 5.5/-6.9 ± 3.4 mmHg in the valsartan group; -9.1 ± 5.9/-6.7 ± 3.8 in the ramipril group; all p<0.01 vs Aml/HCTZ combination). LV mass index was reduced significantly in both treatment groups (-9.6 ± 3.6 g/m
2
in the valsartan group and -7.1 ± 3.1 g/m
2
in the ramipril group; p<0.001 vs amlodipine/HCTZ combination), however the reduction was significantly greater with valsartan than with ramipril (p<0.01). Safety and tolerability were similar across both treatment groups.

Conclusions:
Val/Aml/HCTZ combination was effective in promoving LV mass regression, and such regression was significantly greater than that obtained with Ram/Aml/HCTZ, independent of BP lowering. This finding suggests that valsartan is more effective than ramipril in attenuating this measure of myocardial damage in diabetic hypertensive patients with LV hypertrophy.
Abstract 348: Urinary CYP Eicosanoid Excretion Correlates with Glomerular Filtration in African-Americans with Chronic Kidney Disease
Authors: Dreisbach Albert , Smith Stanley V , Kyle Patrick B , Ramaiah Manjunath , Garrett Michael R , Amenuke Margaret , Griswold Michael E , Roman Richard J
Published: March 2022

Previous studies have indicated that cytochrome P450 (CYP) metabolites of arachidonic acid, ie, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) play an important role in the regulation of renal tubular function and vascular tone. More recent studies suggest that variants in the
CYP4A11
and
CYP4F2
genes are linked to the development of hypertension in a variety of human population studies. However, little is known about the role of 20-HETE or EETs in the pathogenesis of hypertension or diabetic induced renal disease because CYP eicosanoids have not been measured in patients with CKD. The present study profiled HETEs and the dihydroxy metabolites of EETs (DHETs) levels, from spot urines at the time of their clinic visit using LC/MS/MS in 106 African-American patients from the University of Mississippi (UMC) Chronic Kidney Disease (CKD) Clinic with various etiologies of renal disease. Informed consent was obtained for this UMMC IRB approved protocol. Significant positive correlations were found between urinary 5,6- DHETE, 8,9-DHETE, 11,12-DIHETE, 14,15-DHETE, 20-HETE and 19-, 15- and 8-HETE levels and estimated GFR (eGFR) as derived from the MDRD. The magnitude of the influence of urinary eicosanoid levels on eGFR was relatively profound since the slopes of these relationships indicated that there is a 5-10% decrement eGFR associated with each ng/ml fall in urinary eicosanoid levels. These results suggest that a decline in the renal formation of 20-HETE and/or EETs may contribute to the pathogenesis of CKD. At very least, urinary CYP eicosanoids may serve as a useful biomarker for progressive disease.
Abstract 353: Rapid Gene Editing in Rat Models of Human Disease Using Targeted Nucleases
Authors: Geurts Aron M , Schilling Rebecca , Klotz Jason , Kalloway Shawn , Foeckler Jaime , Grzybowski Michael , Hansen Colin , Casati Marc , Richter Samantha , Lazar Jozef , Dwinell Melinda , Moreno Carol , Cowley Allen W , Lombard Julian , Mattson David , Jacob Howard J
Published: March 2022

Previous investigations into mechanisms of genes in animal models have primarily used the mouse because of the availability of technologies for targeted manipulation of its genome in embryonic stem (ES) cells. To accelerate functional mechanistic studies in other species where ES technology is not yet widespread, we recently developed an alternative and rapid method for generating targeted mutations in rat genes by the application of Zinc Finger Nucleases (ZFNs). When introduced into an embryo, ZFNs target and induce a chromosome double strand DNA break at the investigator-specified locus, stimulating cellular responses which can result in knock out or knock in modifications at the locus. Using commercially available reagents, we targeted and disrupted more than 100 protein-coding genes across 13 different inbred, consomic, and outbred genetic backgrounds with a 97% (102/105) success rate in approximately 30 months. ZFNs can therefore access nearly every gene in the rat genome and can induce multiple different alleles at the target locus. The majority of mutations, 81% (355/437) were simple deletions ranging from 1-228 base pairs (median 13-bp) and upon breeding, 94% (178/189) of mutations were transmitted to the next generation with little evidence of germline mosaicism. Many of these strains are now available for functional mechanistic studies of candidate human disease genes for hypertension and chronic kidney disease. Co-introduction of a homologous gene template with the ZFNs could stimulate knockin at three different loci in the rat genome. Gene knock into the rat Rosa26 locus allows for uniform expression of a gene in all cells and tissues of the adult animal as well as germline-competent ES cells derived from the Fawn Hooded Hypertensive strain. This knockin technology in embryos and ES cells offers many new opportunities to make very precise modifications to the rat genome and is a key step toward creating conditional gene alleles in the rat. Transcriptional activator-like effector nucleases (TALENs) are also active in the rat embryo and we have disrupted 70% (7/10) genes with this new technology. In sum, targeted nuclease technology is enabling many new genetic approaches to the rat and other model systems to create even better human disease models.
Abstract 359: Hemodynamic Differences in Normal and Stress Conditions between SJL/J and C3HeB/FeJ Inbred Mouse Strains
Authors: Ilyushkina Irina A , Dyachenko Igor A , Murashev Arkady N , Korshunov Vyacheslav A
Published: March 2022

Introduction:
SJL/J (SJL) mice have elevated (80beats/min) heart rate (HR) compared to C3HeB/FeJ (C3HeB) mouse strain then measured by tail-cuff plethysmography. SJL and C3HeB are differed in behavioral phenotypes. The goal of this study was to investigate hemodynamic responses to normal and stress conditions between SJL and C3HeB inbred mouse strains.

Methods and Results:
Radio telemetry was used for continuous recordings of HR and systolic blood pressure (SBP) in C3HeB (n=5-10) and SJL (n=4-10) mice. We found that both strains showed normal circadian rhythms with little differences in HR and SBP. However, a locomotor activity (LA) of SJL was lower compared to C3HeB (7.8±0.4 vs. 10.0±0.1counts) during the lights off period. A correlation between LA and HR was reduced in SJL (R
2
=0.27) compared to C3HeB (R
2
=0.67) during the lights off period. After a mild stress (transfer to a cage) SJL had a slight HR elevation (70 beats/min) compared to C3HeB in first 5min. LA and SBP were similar between inbred strains after a novel cage test. The HR changes were significantly higher (2.5-fold, p<0.05) in SJL vs. C3HeB during first 5min (30min of restraining period) on the first day of tail-cuff training. Five days of tail-cuff procedures resulted in 2-fold elevation of HR in SJL compared to C3HeB for 30min restraining period. Interestingly, changes in SBP were similar between mouse strains on first day, while SBP showed a trend to be 2-fold higher in SJL vs. C3HeB for 30min on day five. Autonomic control of HR was determined by pharmacological blockages in mice. Atropine (1mg/kg, i.p.) caused significantly greater increases in HR in SJL (160+8beats/min, p<0.05) compared to C3HeB (62+11beats/min). Administration of propranolol (4mg/kg, i.p.) resulted in similar HR reductions between these strains.

Conclusions:
We found that SJL mice are susceptible to elevation of hemodynamic parameters after chronic stress. An increase in sympathetic nervous system activity may explain HR differences between SJL and C3HeB inbred strains.

This study was supported in part by funds from Russian Program "Scientific and scientific-pedagogical personnel of innovative Russia" for 2009-2013 GK№14.740.11.0923 and
HL105623
(VAK)
Abstract 378: Alteration of Renal Neprilysin, Angiotensin Converting Enzyme 2 (ACE2) and Nephrin in Streptozotocin (STZ) Diabetic Mice
Authors: Somineni Hari K , Grobe Nadja , Chodavarapu Harshita , Morris Mariana , Elased Khalid M
Published: March 2022

Diabetic nephropathy is one of the major microvascular complications of diabetes which eventually manifests into end stage renal disease. Alteration in renin-angiotensin system (RAS) is considered to be the primary cause underlying this implication. Emerging evidence suggests that the biological actions of Ang II may be opposed by the formation of Ang (1-7), partly generated by the actions of ACE2 and neprilysin (NEP). NEP is a zinc-containing metallopeptidase catalyzing the conversion of Ang I to Ang (1-7), a potent vasodilator, thus counteracting the deleterious effects of Ang II. In our previous studies it has been shown that renal ACE2 is up-regulated and NEP is down-regulated in type II diabetic (db/db) mice. However data is limited regarding the presence and activity of renal NEP and ACE2 in type I diabetic mice. The goal of the present study is to explore the role of renal NEP and ACE2 in the pathogenesis of diabetic nephropathy in the STZ mouse model of type I diabetes. Diabetes was induced by five consecutive injections of STZ (50 mg/kg, i.p.). Urine was collected in presence of protease inhibitor for measuring albumin, creatinine and total protein contents. STZ diabetic mice exhibit hyperglycemia (305±22 vs 125±6 mg/dL, p<0.001), microalbuminuria (212±34 vs 67±18 μg/day, p<0.05) and renal hypertrophy. Renal ACE2 activity was found to be increased in STZ treated mice (p<0.0001 vs wild type) using the fluorogenic test assay. Renal NEP activity was assessed in situ using a newly established mass spectrometric imaging approach in tissue incubations with the natural substrate, Ang I (m/z1296). As detected via formation of Ang (1-7) (m/z 899), NEP enzyme activity was localized to the renal cortex and significantly reduced by 30% in STZ treated mice (p<0.0001 vs wild type).Western blot analysis demonstrated the expression of renal NEP, ACE2 and nephrin. Immunohistochemical staining illustrated down-regulation of renal NEP, nephrin and up-regulation of medullary ACE2 protein expression in STZ diabetic mice. In conclusion, in spite of increased ACE2, decreased NEP coupled with depletion of nephrin in diabetes could possibly play a crucial role in the development of diabetic nephropathy.
Abstract 409: Endoplasmic Reticulum Stress Mediates Fluid Balance and Metabolic Effects of the Brain Renin-Angiotensin System
Authors: Grobe Justin L , Hilzendeger Aline M , Davis Deborah R , Mark Allyn L , Davisson Robin L , Sigmund Curt D
Published: March 2022

Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) have been identified as important contributors to various neural diseases. We examined the role of ER stress and the UPR in the metabolic and fluid balance effects of brain angiotensin in two mouse models. The ER stress-reducing chemical chaperone tauroursodeoxycholic acid (TUDCA, 5.28 ug/day) or aCSF vehicle was infused into the lateral cerebral ventricle (ICV) of either C57Bl/6J mice treated for three weeks with high dietary sodium and chronic subcutaneous deoxycorticosterone acetate (the “DOCA-salt” model), or transgenic “sRA” mice that express human renin in neurons via the synapsin promoter and human angiotensinogen via its own promoter. Both the DOCA-salt and sRA models exhibit hyperactivity of the brain RAS, suppression of circulating RAS, hypertension, polydipsia, and an elevated resting metabolic rate. Forebrain, midbrain, and hindbrain regions of sRA mice exhibited a significant elevation of CHOP mRNA, a marker of long-term UPR. There were no changes in CHOP expression in the cortex and cerebellum. ICV TUDCA significantly attenuated the polydipsia (DOCA-aCSF 20.7±0.9 vs DOCA-TUDCA 10.8±1.0 mL/day, P<0.05) and metabolic rate (DOCA-aCSF, 3.38±0.07 vs DOCA-TUDCA 3.16±0.06 mL O
2
/100g/min, P<0.05) in the DOCA-salt model. ICV TUDCA had similar effects on the polydipsia in the sRA model (51±10% of aCSF control, P<0.05). Metabolic studies in the sRA model, and blood pressure in the sRA and DOCA models are ongoing. Together these data mechanistically implicate ER stress in the fluid and metabolic responses to increased brain RAS activity.
Abstract 429: Reactive Oxygen Species Impair Endothelial Function and Perivascular Adipose Tissue Signaling in Mesenteric Arterioles From Ovariectomized Rats
Authors: Wang Dan , Zeng Wei , Welch William J , Sandberg Kathryn , Wilcox Christopher S
Published: March 2022

Perivascular adipose tissue (PVAT) normally promotes vascular endothelium dependent relaxation (EDRs), whereas reactive oxygen species (ROS) impair endothelial function. However, the effects of the menopause on PVAT, and its interaction with ROS, are unexplored. We tested the hypothesis that ovariectomy (OVX) causes oxidative stress that impairs microvascular PVAT signaling. Mesenteric arterioles (MAs) were isolated from rats 4-6 weeks after sham-operation (Sham) or OVX. Alternate rats received vehicle or tempol (2mmol · 1
-1
of water) throughout (n=6/group). MAs were preconstricted with 10
-7
M norepinephrine for measurement of acetylcholine (ACh)-induced endothelial derived relaxation (EDR), endothelium derived relaxation factor (EDRF) and NO activity (DAF-FM fluorescence) using a Mulvany-Halperin wire myograph and fluorescence RatioMaster system. Other vessels were studied under spontaneous tone with dilator pathways blocked with 10
-4
M L-NAME, 10
-5
M apamine and 10
-6
M charybdotoxin to measure ACh-induced endothelium dependent contraction factor (EDCF) and ROS generation (temp-9AC fluorescence). OVX rats had vascular remodeling (midia: lumen diameter: 4.4± 0.4 vs 1.5 ± 0.3 μm·μm
-1
, P<0.01). Their MAs without PVAT had diminished EDR (48 ±5 vs 73±3%; P<0.05), EDRF (20±4 vs 41±4%; P<0.01) and NO activity (0.4 ± 0.1 vs 0.7± 0.1 units; P<0.05) and developed an EDCF (23±6 vs 5±3%; P<0.05) and ACh-induced ROS (0.5±0.1 vs 0.1±0.0 units; P<0.01). The presence of PVAT surrounding Sham vessels enhanced EDR (90±3 vs 73±3%; P<0.05), EDRF (57±3 vs 41±3%; P<0.05) and NO (1.2±0.01 vs 0.7±0.1 units; P<0.05). All of these beneficial effects of PVAT were lost in OVX rats. Four to six weeks of tempol drinking in OVX rats restored normal intrinsic vascular endothelial function and NO and restored all the beneficial effects of PVAT while preventing microvascular remodeling. In conclusion, OVX rats had microvascular remodeling and severe endothelial dysfunction ascribed to intrinsic vascular effects of ROS that summated with ROS-dependent interruption of a beneficial microvascular PVAT signaling pathway. Thus, therapeutic targets for menopausal vascular dysfunction should be expanded to include ROS and its extravascular actions on PVAT.
Abstract 434: The Epidemic of Hypertension Related Disease and Changing Patterns of Leading Causes of Hospitalization in Patients with Comorbid Hypertension Among US Adults Aged 35 and Older
Authors: Liu Longjian , An Yuan
Published: March 2022

Objective:
To examine the longitudinal trend of hospitalization due to hypertension related diseases and patterns of leading causes of hospitalization in those with comorbid hypertension among US adults aged 35 and older.

Method:
We used sample data (n=4,598,488 thousand hospitalized cases) from 1980-2007 National Hospital Discharge Surveys to test trends of patients with 1st diagnosis of hypertension (HT) related hospitalization, and any of 2nd to 7th diagnosis of HT, and HT associated with 10 primary causes of hospitalization. Diseases were coded using ICD-9-CM criteria. Age-adjusted rates of disease were calculated using US 2000 standard population. Trends of hospitalization due to HT related disease and associations between primary cause of hospitalized diseases and HT as comorbidity were tested using multivariate logistic regression models.

Results:
Crude hospitalization rates due to 1st diagnosis of HT related disease significantly increased from 1.93% (2327295 / 120579000) in 1980 to 2.14% (5091906/237551000) in 2007. The corresponding age-adjusted hospitalization rates were 1.81% in 1980 and 2.17% in 2007 (test for trend, p<0.01). Similarly, age-adjusted rates due to any of 2nd to 7th diagnosis of HT related diseases significantly increased from 1.23% in 1980 to 5.43% in 2007 (test for trend, p<0.001). Multivariate adjusted logistic regression analysis indicates that in 2007, patients who had any of 2nd to 7th diagnosis of HT, and had primary diagnosis of renal disease had the highest significant odds ratio (OR=7.0, 95%CI: 6.70-7.32) for hospitalization, followed by diabetes (3.47, 3.34-3.59), atherosclerosis (3.10, 2.89-3.32), septicemia (2.08, 1.98-2.19), stroke (1.88, 1.83-1.92), CHD (1.85, 1.81-1.90), heart failure (1.16, 1.12-1.20), and pneumonia and influenza (1.21, 1.16-1.25) than those without these primarily diagnosed diseases, respectively.

Conclusion:
The study, using a nationally representative dataset, is the first to demonstrate a 28-years trend that hospitalization due to HT related diseases significantly increased from 1980 to 2007 in the US adults. The study further highlights that patients with primary diagnosis of renal disease or diabetes with comorbid HT have emerged as top causes of hospitalization.
Abstract 481: Exosomes as a Potential Acellular Signaling Mechanism in the Kidney
Authors: Carlson Julia M , Gildea John J , Pettigrew Abigail C , Courtner Crystal E , Felder Robin A
Published: March 2022

Exosomes are small 50-90 nm vesicles containing mRNA, proteins, and other renal epithelial sub-cellular components and might serve as an intra-nephron signaling mechanism. Exosomes were isolated from cultured immortalized human renal proximal tubule cells (RPTC) by ultracentrifugation following agonist treatment. 24 hour stimulation with the dopaminergic agonist fenoldopam (1 μM) produced a 2.38±0.053 fold increase in exosome release (RFU Vehicle = 0.038±0.0072; n=15, p<0.04). Angiotensin II stimulation (1 nM, 24 hours) caused a 4.71±0.35 fold increase in exosome release (n=18, p<0.04). Agonist-induced responses persisted after removal of extracellular calcium, indicating that renal exosome release is not dependent upon a calcium influx into cells. We next showed that exosomes are able to transfer between cells by electroporating Lamp1-RFP or CD82-YFP (two proteins previously found to be contained in exosomes) into RPTC. We found that both GFP fusion proteins were secreted into the culture medium and taken up by non-transfected cells. We further hypothesized that sodium regulatory proteins would be secreted into exosomes. Proteomic analysis demonstrated the presence of the dopamine-1 receptor and the dopamine-1 receptor selective kinase GRK4, which are known to affect natriuresis, in exosomes derived from RPTC. Furthermore, GRK4 mRNA was detected in exosomes by RT-PCR. In conclusion, exosomes contain proteins and mRNA which could serve as an intra-nephron acellular signaling mechanism that may alter sodium homeostasis.
Abstract 521: Role of Autophagy in Endothelial Cells for Atherosclerosis
Authors: Torisu Kumiko , Torisu Takehiro , Finkel Toren
Published: March 2022

Endothelial dysfunction is thought to play an important role for the development of atherosclerosis. Autophagy is the mechanism by which organelles, aggregated protein, and even lipid droplet are broken down. Since hyperlipidemia can cause endothelial cell dysfunction during the early stage of atherosclerosis, there may be link between atherosclerosis and endothelial autophagy. To address this issue, we generated endothelial specific Atg7 knockout in apoE deficient mice (Atg7
endo
/apoE).
En face
analysis of Oil-red O stained whole aorta showed Atg7
endo
/apoE double knockout mice had severe atherosclerosis. At 16 week old, apoE single knockout mice (apoE) had 9.7 % of oil-red O positive area while Atg7
endo
/apoE mice had 18.6 % (n=10, 9 mice per group). The composition of macrophages, collagen, and lipid in atherosclerotic lesions were similar between control and Atg7
endo
/apoE mice. Body weight and fat composition were also similar between the two groups, as were blood glucose, triglyceride, cholesterol, and fatty acid levels. At 24 weeks of age,
en face
analysis of aorta showed even more disparity in lesion size between control and Atg7
endo
/apoE mice (apoE mice, 15.4%, Atg7
endo
/apoE mice, 36.2%, n=10, 7 mice per group). Secretion of various chemokine, such as MCP-1 from Atg7 knockdown human umbilical vein endothelial cell (shATG7 HUVECs) was 10-fold higher that of control cells. These results suggest that autophagy deficiency in endothelial cells accelerate in vivo atherosclerosis. This augmentation may result from an enhanced inflammatory response.
Abstract 53: Telmisartan But Not Tempol Improves the Already Established Renal Cell Senescence in Type 2 Diabetic Rats
Authors: Nakano Daisuke , Kitada Kento , Ohsaki Hiroyuki , Hitomi Hirofumi , Kobori Hiroyuki , Imanishi Masahito , Nishiyama Akira
Published: March 2022

We previously reported that hyperglycemia induces renal tubular cell senescence in type 1 diabetic animals. In the present study, we evaluated the tubular cell senescence in type 2 diabetic animals and investigated whether the renal cell senescence could regress in response to treatment with a reno-protective dosage of an angiotensin receptor blocker, telmisartan (10 mg/kg/day). Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of spontaneously developed type 2 diabetes, showed an age-dependent increase in renal cell senescence analyzed by p21 expression (6 weeks old: 1.3±0.3 fold, 22 weeks old: 2.6±0.3 fold, vs. age-matched lean control rats, n=8, respectively) and senescence-associated β galactosidase (SABG) staining. OLETF rats were separated into the following 3 groups at 22 weeks of age and were kept under treatment until 34 weeks of age (n=10 per group): 1) un-treated, 2) telmisartan, and 3) tempol (an anti-oxidant, 3 mmol/L in drinking water). Neither treatment affected the blood glucose levels. Telmisartan, but not tempol, markedly reduced SABG staining by 34 weeks of age compared with that at 22 weeks of age or with that of age-matched un-treated rats. Increase in the expression of p21 was significantly smaller in telmisartan-treated rats (1.3±0.2 fold vs. lean rats) than in un-treated rats (2.9±0.2 fold, p<0.01) and tempol-treated rats (3.0±0.2 fold, p<0.01). Un-treated OLETF rats showed a greater intrarenal expression of TNF-α than lean rats, and the increase was suppressed by telmisartan. On the other hand, ED-1-positive macrophages rarely colocalized with p21-positive tubules at any stage of diabetes in any treated groups. These results indicate that type 2 diabetes prompts the renal cell senescence and that accumulation of renal senescent cells is reversible. Macrophage infiltration seems unlikely to contribute to the reduction of senescent cells. In addition, while there are many reports showing the involvement of oxidative stress in cell senescence, anti-oxidant tempol did not affect the established cell senescence.
Abstract 530: Vascular Remodeling by Angiotensin Peptide Hormones Alters Reactivity of the Microcirculation
Authors: Carver Kyle A , Smith Thomas L , Gallagher Patricia E , Tallant E. Ann
Published: March 2022

Endothelial dysfunction associated with hypertension leads to end-organ damage through impairment in tissue perfusion. Angiotensin II (Ang II) causes vascular remodeling and fibrotic stiffening and attenuates endothelial-dependent vasorelaxation. However, the heptapeptide hormone angiotensin-(1-7) [Ang-(1-7)] reduces the structural effects of Ang II and acutely vasodilates vessels
ex vivo
. Lewis rats were chronically treated with either Ang II, Ang-(1-7), or both angiotensin peptides to examine the effect of the peptide hormones on microvascular remodeling and reactivity of cremaster muscle arterioles
in vivo
. Cremaster arterioles (50 microns in diameter) were visualized by intravital microscopy to measure vascular reactivity. Ang II elevated systolic blood pressure by 43 mmHg, an effect that was not changed by co-administration of Ang-(1-7); Ang-(1-7) alone had no effect. Ang II increased the media to lumen ratio (by 82% compared to Sham, n=7, p<0.01) and perivascular fibrosis (by 133% compared to Sham, n=7, p<0.001), while co-treatment with Ang-(1-7) attenuated the Ang II-enhanced remodeling and fibrosis to values no different than Sham or Ang-(1-7) alone. Phenylephrine (PE) elicited vasoconstriction in Sham rats, reducing vessel diameter by 21%, and this effect was not altered in rats treated with Ang II. Although PE-constricted arterioles from Sham rats vasodilated in response to acetylcholine (Ach, a 31.4% increase in diameter), vessels from Ang II-treated rats had a blunted response to Ach (a 9% increase). These results suggest that the Ang II-mediated microvascular remodeling altered microvascular vasodilation. In contrast, arterioles from rats treated with Ang-(1-7) or the combination of Ang-(1-7) and Ang II did not constrict in response to PE or endothelin. However, acute administration of the nitric oxide synthase inhibitor L-NAME to Ang-(1-7)-treated rats restored the vasoconstrictive response to PE, indicating that Ang-(1-7) increases endogenous nitric oxide to maximally dilate microvascular arterioles and prevent constriction. These results suggest Ang-(1-7) may serve as an effective adjuvant therapy to attenuate end-organ damage by increasing blood flow and tissue perfusion caused by hypertension.
Abstract 606: DPP-4 Inhibition Improves Diastolic dysfunction in obese mice fed a Western Diet
Authors: Nistala Ravi , Habibi Javad , Johnson Megan , Garro Mona , Aroor Annayya , Whaley-Connell Adam , DeMarco Vincent , Sowers James R
Published: March 2022

Background and rationale:
Obesity and diabetes are associated with insulin resistance, salt sensitive hypertension and cardiovascular dysfunction. Pharmacological inhibition of dipeptidylpeptidase-4 (DPP-4) to increase the bioavailability of glucagon like peptide-1 (GLP-1) is a contemporary therapy for control of glycemia in Type2 diabetes patients. Whether DPP-4 inhibition lowers blood pressures and improves pre-diabetic cardiomyopathy in a diet induced obesity (DIO) mouse model has not been examined in detail.

Methods:
C57Bl/6 mice were fed high fat/high sucrose (Western diet, WD) for 16wks starting at ∼4 wks of age and treated with MK-0626 (10mg/kg/day, Merck).

Results:
When compared to normal chow-fed mice, Western diet-fed (DIO) mice manifested increased MAP (15-20mmHg), heart (50%) and body weight (33%), increased blood sugars (FBS) and sodium retention (urine sodium 6.9μmol/day/gBW vs. 2.3μmol/day/gBW, respectively). In addition to correcting FBS, MK-0626 lowered MAP and increased sodium excretion and corrected cardiac diastolic dysfunction (
Table
). Indeed, echocardiographic indices of diastolic function, including tissue doppler E’/A’ ratio, estimates of LV filling pressure (E/E’), isovolemic relaxation time and the Tei index, all abnormal in mice fed a WD, were improved by MK-0626. Contemporaneous with these cardiac functional abnormalities, the mTOR/S6K and ERK1/2 growth signaling pathways were activated in DIO mice and improved by 16 wks of MK-0626 treatment.

Conclusion:
These studies suggest that MK-0626 increases cardiac and renal bioavailability of GLP-1 and improves myocardial GLP-1R signaling, myocardial remodeling resulting in increased renal sodium excretion and improvements in cardiac diastolic function in pre-diabetic insulin resistant DIO mice.

Table:
Summary of 2D M-Mode, pulse wave and tissue doppler Echo measurements on 20 wk old mice on control diet (CD), Western diet (WD), or Western Diet with MK-0626 (WD-MK). Values are mean ± SE. Numbers in parentheses are sample sizes. Paired t-tests: *p<0.05 CD vs WDC; † P<0.05 WDC vs WD-MKE, velocity of early mitral flow; E’, peak velocity of septal annulus; E/E’ index of LA filling pressure; Vp, flow propogation velocity; E/Vp, index of LV filling pressure; IVRT, isovolumic relaxation time; MPI, myocardial performance index.
Abstract 621: The G Protein-Coupled Estrogen Receptor GPER is Required for Induction of Nox Activity in Mice
Authors: Meyer Matthias R , Fredette Natalie C , Prossnitz Eric R
Published: March 2022

The Nox family of NADPH oxidases is a major source of reactive oxygen species (ROS) such as superoxide (O
2
-
) and hydrogen peroxide (H
2
O
2
), which importantly influence cardiovascular function. Vasoactive agonists such as angiotensin II can stimulate Nox-dependent ROS production, whereas endogenous estrogens act as antioxidants under certain conditions. We hypothesized that the intracellular transmembrane G protein-coupled estrogen receptor GPER is involved in the regulation of Nox activity. Using GPER
-/-
and WT mice, we determined the effect of angiotensin II on O
2
-
production in isolated vascular smooth muscle cells (VSMC) by lucigenin-enhanced chemiluminescence, and on contractile responses in renal arteries prepared for recording of isometric tension. The contribution of NADPH oxidases was assessed using its specific inhibitor gp91ds-tat (3 μmol/L). Angiotensin II (100 nmol/L) acutely increased O
2
-
production in WT VSMC, an effect blocked by gp91ds-tat (6.6±2.1 AU
vs.
-0.8±2.2 AU after 20 min,
n
=4-5,
p
<0.05). Surprisingly, angiotensin II was unable to induce O
2
-
formation in GPER
-/-
VSMC (-0.2±1.0 AU
vs.
6.6±2.1 AU,
n
=5,
p
<0.05
vs.
WT). Following administration of NADPH (100 μmol/L), maximal NADPH oxidase activity in GPER
-/-
VSMC was also reduced by 73% (442±185 AU
vs.
1617±468 AU,
n
=5,
p
<0.05
vs.
WT), indicating that the induction of Nox activity is largely impaired by GPER deficiency. In line with these findings, contraction to angiotensin II was 1.9-fold less potent in GPER
-/-
renal arteries (35±4%
vs.
66±11% of contraction to 60 mmol/L KCl,
n
=5,
p
<0.05
vs.
WT); this difference was abolished by gp91ds-tat, which decreased the contractile response in WT by 47% (
p
<0.05), but had no effect in GPER
-/-
arteries. In conclusion, these data indicate that GPER is required for induction of vascular Nox activity in mice, and suggest that estrogen-dependent regulation of ROS production may not be limited to antioxidative effects.
Abstract 643: Gender -based Differences In Left Ventricular Remodeling After Myocardial Infarction: Insight Into The Protective Role Of Integrin-linked Kinase
Authors: Manchini Martha T , Sofia Renato R , Serra Andrey J , Antonio Ednei L , Oliveira Fernanda A , Tucci Paulo J , Silva Jose A
Published: March 2022

There is conflicting data about gender differences with regard to left ventricular (LV) remodeling after myocardial infarction (MI). Moreover, the molecular basis for gender-based differences in LV remodeling remains unknown. This study evaluated whether gender differences exist in LV remodeling after MI. Given that integrin-linked kinase (ILK) influences LV remodeling, we also examined whether gender affects the modulation of ILK post-MI. Female and male Wistar rats were assigned to one of three groups: a sham group, a moderate MI group (size: 20-39% of LV area) and a large MI group (size: ≥40% of LV area). The MI was produced by coronary occlusion and echocardiographic analysis as well as ILK expression was performed six weeks post-MI. The MI resulted in systolic dysfunction and enlargement of end-diastolic as well as end-systolic dimension of the left ventricle as a function of necrotic area size for both genders. However, female rats with large MI showed a lower diastolic (5.4±1 mm
2
) and systolic dilatation (3.9±0.9 mm
2
) than respective male rats (8.2±0.7 mm
2
and 5.7±1.1 mm
2
, respectively). Both gene (moderate MI: 1.85±0.15; large MI: 2.35±0.16, p<0.001) and protein (moderate MI: 3.21±0.09; large MI: 4.75±0.24, p<0.001) levels of ILK were increased as a function of MI size in female rats. However, only male rats in the large MI group showed an altered ILK mRNA level (moderate MI: 1.51±0.10; large MI: 1.69±0.08, p<0.001). A negative linear correlation was evident between LV dimensions and ILK protein expression in female rats with large MI (left ventricle diastolic area:ILK protein content: r
2
=0.52, p=0.003; left ventricle systolic area:ILK protein content: r
2
=0.68, p<0.001). In conclusion, post-MI ILK expression is altered in a gender-specific manner and higher ILK levels found in females may be sufficient to improve geometry of LV; however, may not be sufficient to improve LV function.
Abstract 644: Tetrahydrobiopterin Supplementation Increases Renal Inner Medullary Nitric Oxide Synthase Enzymatic Activity in Male, But Not Female Spontaneously Hypertensive Rats
Authors: Brinson Krystal N , Sullivan Jennifer C
Published: March 2022

Tetrahydrobiopterin Supplementation Increases Renal Inner Medullary Nitric Oxide Synthase Enzymatic Activity in Male, But Not Female Spontaneously Hypertensive Rats

Krystal N. Brinson and Jennifer C. Sullivan, Experimental Medicine, Georgia Health Sciences University, Augusta, GA 30912

We previously published that male spontaneously hypertensive rats (SHR) having less nitric oxide synthase (NOS) enzymatic activity in the renal inner medullary (IM) than female SHR, which cannot be explained by protein expression alone. Tetrahydrobiopterin (BH4) is an essential cofactor for NOS activity. BH4 is easily oxidized to BH2, which is not a NOS cofactor, and male SHR have greater levels of oxidative stress than females. Therefore, we hypothesized that 1) male SHR will have greater increases in NOS enzymatic activity following BH4 supplementation than females and 2) antioxidant treatment will result in greater increases in NOS enzymatic activity in male SHR regardless of BH4 supplementation. Studies measured renal IM NOS enzymatic activity in the presence and absence of BH4 from 12-13 week old control (N=5-6) or antioxidant treated (tempol, 30 mg/kg/day in drinking water) male and female SHR (N=4-5). Supplementation with BH4 increased NOS activity in male SHR (177±35 vs. 315±52 pmol/mg; p=0.04), with no effect in females (434±112 vs. 548±84 pmol/mg). Following 2 weeks of antioxidant treatment, NOS enzymatic activity in male SHR was significantly increased compared to control SHR (p=0.04), and supplementation with BH4 no longer altered NOS enzymatic activity in males (588±143 vs. 648±153 pmol/mg). Antioxidant treatment did not significantly alter renal IM NOS enzymatic activity in female SHR compared to untreated controls, and BH4 supplementation did not alter NOS activity in tempol-treated females (473±82 vs. 580±66 pmol/mg). Tempol treatment also abolished the sex difference in renal IM NOS enzymatic activity. In conclusion, our data suggest that greater levels of oxidative stress in male SHR contributes to lower levels of NOS enzymatic activity compared to females. We hypothesize that this results in greater BH4 deficiency in males contributing to decreases in NO and increases in blood pressure.