bluuebunny/crossref_metadata_2025 huggingface dataset:
Neonicotinoid insecticide imidacloprid impairs lipid metabolism in 3T3‐L1 cells
Authors: Yoon Kyong Sup , Kim Younghwa , Seo Seulgi , Lee Junsoo , Park Yeonhwa , Park Yooheon
Published: June 2021

Exposures to environmental pollutants including several classes of insecticides have been positively or negatively linked to obesity epidemic. Imidacloprid, a relatively new insecticide belongs to a class called neonicotinoid, has been increasingly used to control agricultural insect pests, structural pests, and ectoparasites significantly affecting human and animal health. Although medical and public interests towards the environmental exposure to insecticides are growing, information about imidacloprid exposure affecting human and animal health is very limited. To elucidate mechanism of impairing lipid metabolism, we tested the effect of imidacloprid on adipogenesis in 3T3‐L1 cells. 3T3‐L1 pre‐adipocytes were differentiated by the addition of insulin (1μg/ml), dexamethasone (1μM), IBMX (10mM), and 10% FBS in DMEM medium. Cells were treated with or without imidacloprid (10 and 20μM). No cytotoxicity was observed in concentration of 20μM imidacloprid for 6 days of differentiation as reported elsewhere. Lipid accumulation was measured by Oil‐red O staining and intracellular triacylglyceride contents were analyzed. Level of mRNA and protein expression of the lipid metabolism related transcription factors were measured by RT‐PCR and Western Blot analysis, respectively. Imidacloprid significantly increased adipocyte differentiation as well as lipid accumulation in 3T3‐L1 cells.
Production of novel platycodin D derivatives by Aspergilli
Authors: Ahn Hyung Jin , You Hyun Ju , Suh Joo‐Won , Ji Geun Eog
Published: June 2021

Platycodin D (PD) is the major saponin components abundant in the root of Platycodon grandiflorum, which has been reported to have anti‐inflammatory and anti‐tumor activities. PD contains sugar residues including glucose unit at C‐3 and apiose‐xylose‐rhamnose‐arabinose residue at C‐28. In the present study, 8 noble PD derivatives with diverse sugar chains were produced by the crude enzyme extracts of Aspergillus niger and A. usamii grown in the presence of various carbon sources. Commercially available enzymes such as hesperidinase, β‐glucosidase, and β‐xylosidase were not effective in cleaving the glycosidic side chains of PD. Depending on the sugar side chains the PD derivatives demonstrated different degree of toxicity against a normal fetal colon cell line. These findings could be informative to obtain novel PD derivatives with reduced cytotoxicity by Aspergilli. This work was supported by Industrialization Support Program for Bio‐technology of Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (810007‐03‐1‐WT211), Republic of Korea.
Room temperature stabilization of gene transcripts and genomic DNA in whole blood
Authors: Steven P , Wilkinson  , Stassinopoulos Angela , Whitney Scott , Ohgi Sharron , Clement Omo , Muller‐Cohn Judy , Muller Rolf
Published: June 2021

Genomic and mitochondrial DNA and gene expression profiles from blood samples are increasingly being used to diagnose specific diseases, monitor disease progression and assess patient responses to medical treatment. However, such applications require reliable preservation of total RNA and DNA in patient samples during collection, transport and storage. Numerous agents threaten DNA and RNA profiles in collected blood samples, including nucleolytic attack, oxidative damage and hydrolysis. Such damage can severely disrupt genotyping and DNA sequencing efforts. Transcription profiles are highly dynamic and can change rapidly during and after blood collection using current methods, potentially affecting interpretation of the expression analysis. In this study, we compare DNAgard Blood, PAXgene Blood DNA and freezer storage in their capacities to preserve genomic DNA integrity in human whole blood for 6 months. In a separate analysis, we compare RNA stabilization properties of PAXgene Blood RNA, freezer storage and a novel stabilization formulation by assessing changes in the relative expression of a panel of genes in whole blood specimens over the course of 7 days using qRT‐PCR and the Human Common Cytokines PCR Array (SABiosciences). Our results demonstrate the importance of selecting an appropriate blood storage method to obtain reliable gene expression and genomic data.
Time‐dependent expression of Endothelin‐1 in lungs and the effects of TNF‐ƒÑ blocking peptide on acute lung injury in an endotoxemic rat model
Authors: Jesmin Subrina , Mowa Chishimba , Yamaguchi Naoto , Zaedi Sohel , Sultana Sayeeda Nusrat , Gando Satoshi
Published: June 2021

Endothelin (ET)‐1 is a potent vasoconstrictor that has been implicated in the pathogenesis of a number of diseases, and some studies suggest that circulating ET‐1 is elevated in sepsis. The present study investigated whether ET plays a role in sepsis‐mediated acute lung injury and whether its expression (ET) could be down regulated by blockade of TNF‐α in septic lung. Male Wistar rats at 8 weeks of age were administered with either saline or lipopolysaccharide (LPS) at different time points (1, 3, 6 and 10 hours), after which various tests were then performed. The features of acute lung injury were observed at 1h after LPS administration, which gradually became severe with time. Systolic and diastolic pressures were reduced just about an hour after LPS administration, where as pulmonary TNF‐α levels were significantly increased at various time points after LPS administration. LPS induced a time‐dependent expression of ET‐1 and ETA receptor in the lungs compared to control, peaking and increasing by 3 fold at 6 h after induction of endotoxemia, whereas levels of ETB receptor, which has vasodilating effects, were remarkably down regulated time‐dependently. We conclude that the time‐dependent increase of ET‐1 and ETA receptor with the down regulation of ETB receptor may play a role in the pathogenesis of acute lung injury in endotoxemia. Finally, treatment of LPS‐administered rats with TNF‐α blocking peptide for three hours significantly suppressed levels of pulmonary ET‐1. These data taken together, led us to conclude that differential alteration in ET expression and its receptors may be mediated by TNFα and may, in part, account for the pathogenesis of acute lung injury in endotoxemia.
An algorim for quantification of hemodynamic properties in murine dorsal window chamber video images
Authors: Fontanella Andrew Nicholas , Palmer Greg , Schroeder Thies , Dewhirst Mark W
Published: June 2021

We have developed a computational algorithm for the quantification of hemodynamic parameters from video‐rate images of blood flow in a dorsal window chamber model. This method analyses local correlations in pixel intensity fluctuations due to single erythrocyte movement to determine flow rates and direction of movement. The ability to quantify these parameters is of major importance in assessing vascular remodeling in tumor models. Since vascular oxygen/nutrient/drug transport dynamics play a major role in determining tumor progression and response to treatment, the ability to automatically quantify treatment induced effects is an invaluable asset to the dorsal window chamber model. Whereas earlier efforts to quantify flow dynamics relied heavily upon manual vessel identification and sampling of segments of interest, this method provides an entirely automated method by which values for relative velocity and direction of movement can be assigned to every pixel in the field of view. This advancement in analysis will facilitate much more rapid and less biased data generation than currently available image analysis techniques.
Angiotensin II induced IL‐6 m RNA and cell growth through JAK2 in Rat Cerebellar Astrocytes
Authors: Kandalam Umadevi , Clark Michelle A
Published: June 2021

The pleiotrophic effects of angiotensin II (Ang II) play important roles in astrocyte growth and inflammatory responses. We investigated whether Ang II induces astrocyte growth and interlukin‐6 (IL‐6) mRNA levels in rat cerebellar astrocytes through Janus Kinase 2 (JAK2). Ang II increased JAK2 phosphorylation in a time‐ and a dose‐dependent manner. Maximal phosphorylation of JAK2 was observed with 100nM Ang II at 15 minutes. The phosphorylation of JAK2 was blocked by AG490, a JAK2 inhibitor. Losartan, a selective AT1 receptor antagonist, inhibited Ang II‐mediated JAK2 phosphorylation, while pretreatment with an AT2 receptor blocker was ineffective. Ang II increased the mRNA expression of IL‐6 in a concentration‐and time‐dependent manner. Maximal IL‐6 mRNA expression occurred with 100 nM Ang II, and the peak effect occurred at 3 hours. Moreover, treatment with AG490 prevented Ang II‐induced IL‐6 mRNA levels, and Ang II‐induced astrocytes growth. The present study has demonstrated that Ang II induced the phosphorylation of JAK2 via the AT1 receptor in cerebellar astrocytes. In addition, our results suggest that JAK2 is an upstream signal that mediates Ang II‐induced IL‐6 expression and astrocyte growth. These findings represent a novel nonclassical mechanism of Ang II signaling in cerebellar astrocytes. (Support NHLBL HLO77199 and NSU PFDRG).
Olmesartan reduces angiotensin II without affecting ACE activity in kidneys of mRen2.Lewis rats
Authors: Varagic Jasmina , Ahmad Sarfaraz , Moniwa Norihito , Brosnihan K. Bridget , Ferrario Carlos Maria
Published: June 2021

We investigated the possibility that the antihypertensive actions of the angiotensin II (Ang II) receptor blocker –olmesartan medoxomil– may in part be associated with a functional inhibition of angiotensin converting enzyme (ACE) activity due to increased Ang‐(1–7) production in the absence of changes in plasma Ang II (Ichikawa, 2001; Agata, 2006). Experiments were performed in 10 – 11 week old mRen2.Lewis congenic hypertensive rats administered either a saline vehicle (VEH, n=7) or olmesartan (OLM: 0.5 mg/kg/day for 6 weeks; n=7) by subcutaneous implantation of osmotic minipumps. Plasma and kidney tissue Ang peptides and serum and kidney ACE activity were measured at the completion of the experiments.

VEH
OLM

SAP (mm Hg)
208 ± 3
99 ± 3*

Kidney cortex

Ang peptides (fmol/mg)

Ang I
7.6 ± 0.9
16.7±2.2*

Ang II
15.9±1.6
6.3±0.6*

Ang‐(1–7)
10.3±1.6
7.71±1.5

ACE activity (nmol/mg/min)
1.9±0.7
2.4±0.9

Plasma/Serum

Ang peptides (fmol/ml)

Ang I
92±6
565 ± 77*

Ang II
31±5
254 ± 43*

Ang‐(1–7)
27±4
60 ± 5*

ACE activity (nmol/ml/min)
69±5
77 ± 1

SAP= Systolic Arterial Pressure;

p<0.05

Our results support the concept that the reduction in Ang II kidney content may contribute to the protective effects of olmesartan on renal structure and function. We also provide evidence for independent regulation of Ang II between plasma and kidney tissue in response to AT1 blockade. The absence of changes in ACE activity suggests no measurable effect of olmesartan on ACE activity. This work is supported by an unrestricted grant from Daiichi Sankyo, Inc.
Ferrets ‐ a small animal alternative to dogs in cardiovascular pharmacology studies
Authors: Main Bradley W , Hunter Randy , Adams Stephanie , Doyle Jennifer , Clawson Christine , Gould Kenneth
Published: June 2021

Ferret cardiac ion channels are similar to those in human, dog and to those in species used for in vitro cardiac electrophysiology studies. The ferret heart is relatively large making it suitable for instrumentation while their overall small size (approximately 900g) allows for reduced compound use. The ferret can be a suitable alternative to the dog for early screening of compounds with potential cardiovascular effects. Recent technology improvements in small animal telemetry allow the acquisition of two pressure signals in a single animal. We implanted HD‐S21† pressure transmitters into ferrets and recorded arterial and left ventricular pressures and ECGs in response to milrinone.

Dose responsive increases were observed in left ventricular function including indices of inotropy, lusitropy and heart rate. Systemic arterial blood pressure decreased at 3 and 10 mg/kg of milrinone.

Milrinone Dose
Systolic Pressure* (mmHg)
LV Heart Rate* (beats/min)
dP/dtmax * (mmHg/sec)
dP/dtmin * (mmHg/sec)

1 mg/kg
no change
↑ 51.7 ± 16.2
↑ 6953 ± 1693
↑ 1419 ± 987

3 mg/kg
↓ 23.6 ± 4.8
↑ 92.0 ± 14.7
↑ 8011 ± 1212
↑ 1840 ± 563

10 mg/kg
↓ 26.1 ± 4.9
↑ 114.2 ± 9.7
↑ 8490 ± 661
↑ 2006 ± 880

Change from vehicle‐treated group

In summary, data with a high degree of fidelity and reproducibility can be obtained in a small animal species like the ferret. This species can be used as an alternative to the dog in cardiovascular pharmacology studies.
Atorvastatin protects against aorta contractility impairment in insulin‐resistant rats
Authors: El‐Moselhy Mohamed Ahmed , Mahmoud Mona F. , El‐Bassossy Hany M.
Published: June 2021

This study aimed to investigate the possible protective role of atorvastatin against the vascular dysfunction associated with insulin resistance (IR) in high fructose‐fed rats. In the pursuit of this aim, blood levels of glucose, cholesterol, insulin as well as vascular reactivity and insulin resistance index were assessed in a well‐established model of insulin resistance in presence or absence of atorvastatin (10 mg/kg/day for 8 weeks). Fructose feeding (10% fructose in drinking water for 8 weeks) induced hypercholesterolemia, hyperinsulinemia without any change in blood glucose level. Additionaly, serum tumor necrosis factor (TNF‐α), insulin resistance index, leukocyte infiltration of aorta and endothelial cell pyknosis were elevated. Fructose feeding induced hyperresponsiveness to both phenylephrine (PE) and KCl and hyporesponsiveness to ACh but not to sodium nitroprusside (SNP) relaxations. Most importantly, in atorvastatin‐treated rats, blood levels of cholesterol, insulin and TNF‐α, in addition to insulin resistance index were significantly reduced. Furthermore, atorvastatin treatment reduced leukocyte infiltration and endothelial cell pyknosis and decreased the hyperresponsiveness to both phenylephrine and KCl. In conclusion, the current study highlights a protective role for atorvastatin against the impairment in aortic vascular reactivity via a mechanism that involves reduction of the elevated cholesterol and TNF‐α levels in insulin resistance rats.
The Cost Effectiveness of Topical Minoxidil Therapy in Management of Baldness
Authors: Raja Dewan Saiful , Sultana Bahar
Published: June 2021

“Use it or loss it (Rogaine),” is an advertisement as a cure for baldness. Rogaine is a commercial name for Minoxidil. Minoxidil is a vasodilator used orally for treatment of severe to malignant hypertension. Minoxidil therapy also causes hypertrichosis (increase of body hair). This drug is used topically to treat baldness.

Minoxidil is not effective in halting the receding hairline on the frontal scalp. Its antibald effect is limited to the vertex of the head. It takes several months (2–4) to regrow hair by twice‐a‐day application. Continuous use of Minoxidil topical solution is needed to maintain hair regrowth. Stopping Minoxidil application causes loss of newly regrown hair in 3 to 4 months.

Purpose of this case study will answer several questions. Is Minoxidil effective against baldness? Is it a permanent, cost‐effective remedy for baldness? What age group will get the maximum benefit from Minoxidil therapy? What types of baldness are amenable to Minoxidil therapy? Is it effective in female baldness? Is Rogaine free from remarkable adverse effects? What are the causes of baldness? Is there any permanent remedy for baldness?
Role of vitamin C on protection against toxic effects of cadmium
Authors: Sopi Skender , Masurica Feim , Bozo Seit
Published: June 2021

In the present study, an investigation was undertaken to assess the protective efficacy of vitamin C on cadmium (Cd) toxic effects in liver. New Zeland rabbits were divided in four groups and orally was given Cd (2 mg/kg/bw/day) or ascorbic acid (AA), (50 mg/kg/bw/day) alone, and in one group of animals both ascorbic acid and Cd were applied simultaneously in same animals. Control animals received saline. The activity of plasma hepatic marker enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), superoxide dismutase (SOD) and catalase, as well as membrane lipid peroxidation (LPO) was measured. In Cd treated animals, activity of enzymes and LPO were increased significantly, compared with control animals, however in AA treated group these parameters did not change. Simultaneous treatment of animals with AA and Cd, decreased the enzymes activity and LPO almost at control level. The result of this study, confirm a protective role of vitamin C on Cd‐induced liver damage.
Interactions between Cytochromes P450 2B4 (CYP2B4) and 1A2 (CYP1A2) Lead to Alterations in Toluene Disposition and P450 Uncoupling
Authors: Reed James Robert , Cawley George F. , Backes Wayne L.
Published: June 2021

The goal of this study was to elucidate the effects of CYP1A2 and CYP2B4 interaction on the oxidative metabolism of toluene and the degree of reaction coupling by comparing metabolism by simple lipid reconstituted systems containing P450 reductase (CPR) and a single P450 to that by a mixed system with CPR and both P450s. CYP2B4 generated benzyl alcohol with low levels of p‐cresol, whereas CYP1A2 formed minor amounts of o‐cresol and benzyl alcohol. When both P450s were mixed, the rates of catalysis by CYP2B4 and CYP1A2 were decreased and increased, respectively. Assuming that the efficiency of catalysis by each enzyme was unchanged in the mixed systems, the expected rates of NADPH oxidation and the production of hydrogen peroxide and excess water by the mixed system were calculated. The major effect of the P450‐P450 interaction was to decrease the rate of excess water production. By comparing peroxidative metabolism (using hydrogen peroxide instead of NADPH) and the rates of CPR‐mediated P450 reduction in simple and mixed systems, it was shown the P450‐P450 interaction affected the rate‐limiting step of CYP1A2 catalysis and not necessarily the CPR‐binding affinities of the P450s. (Funding: ES004344)
Hippocampal Neurogenesis in Ames Dwarf Mice
Authors: Andrieux Sybil , Key Mary , Ilenbiluan Onome , Soto Armando , Walters Amy , Hill Tiffany , Watts Brandy , Chan Victor , Morris Mariana
Published: June 2021

Neuronal generation had been thought to occur only during development; however, current research indicates that neurogenesis continues into adulthood in the hippocampus and olfactory regions. The model selected for study was the Ames Dwarf mouse, homozygous for the PROP‐1 gene mutation and deficient in pituitary hormones. Normal‐sized heterozygous litter mates were used as controls. The goal was to determine whether there were alterations in brain neurogenesis using the bromodeoxyuridine (BrdU) method to label dividing cells. 12‐month‐old male Dwarf and control mice (n=5–6/group) were injected IP for seven days with 50mg/kg of BrdU. Animals were perfused with paraformaldehyde, and consecutive 25 um sections were taken through the hippocampus. Immunofluorescence was used to localize BrdU and neuronal nuclear antigen, NeuN, a marker for mature neurons. There was no difference in the number of BrdU‐positive cells in Dwarf mice as compared to controls (13.5 ± 5.1 vs 12.6 ± 2.7). The percentage of co‐labeled BrdU and NeuN neurons showed a trend to be higher in Dwarf mice (69.9 ± 5.9% vs. 61.9 ± 12.6%; Dwarf vs control). Data indicate that there is an increase in proliferation of neural progenitor cells that can develop into mature neurons in the hippocampus of Ames Dwarf mice over controls. Supported by NIH R25GM086257 and Henry Jackson Foundation Subaward #686190.
HEMOLYSED BLOOD‐INDUCED VASOMOTOR DYSFUNTION IN ISOLATED RAT CEREBRAL ARTERIES
Authors: Cseplo Peter , Vamos Zoltan , Koller Akos
Published: June 2021

The underlying vasoconstrictor mechanisms following hemorrhagic stroke have not yet been elucidated. We hypothesized that hemolysed blood induces vasomotor dysfunction in isolated cerebral arteries. The basilar artery (BA) from male rats were isolated, cannulated and placed in a pressure‐myograph organ chamber. The diameters of vessels were measured by videomicroscopy in the presence of physiological intraluminal pressure (80 mmHg) without intraluminal flow. The vasomotor function of the vessels was studied in response to hemolysed blood (HB). Dilatory function of vessels were obtained in control conditions, in the presence of HB and after washout of HB. The basal diameter of BA was 264±7,5 μm, whereas the passive diameter was 404±10 μm, indicating that a substantial myogenic tone developed. HB reduced the basal diameter of BA (to 204±13 μm, 77±4% of AD). After washing out of HB, the diameter of BA was 284±9 μm. In control ACh, SNP and nifedipine elicited substantial dilations (16±2%, 23±3% and 31±2%) of cerebral arteries. In contrast, in the presence of HB dilations to ACh, SNP and nifedipine (12±2%, 16±2%, 21±2%) were significantly decreased. In conclusion, the present findings suggest that hemolysed blood increases the myogenic tone and inhibits both endothelium‐dependent and ‐independent dilator mechanisms of cerebral arteries.
In vitro characterization of a dual‐receptor targeted drug delivery system for treating vascular diseases
Authors: Lamberti Giuseppina , Wu Fan , Kiani Mohammad F. , Wang Bin
Published: June 2021

Introduction

Drug delivery systems targeted to the vasculature provide an effective mean for increasing the therapeutic efficacy of various drugs. In this study we present a novel dual‐receptor (selectins and ICAMs) targeting approach to enhance the drug carrier's binding efficiency to the inflamed tissue in vascular diseases.

Methods

Florescent microspheres were prepared by coating the surface with different ratios of antibodies against ICAM‐1 (aICAM‐1) and E‐selectin (aE‐selectin). A flow chamber was used to study the interaction of the microspheres with TNF‐α activated HUVEC under different shear flow conditions.

Results

Microspheres coated with 2:1 ratio of aICAM‐1 and aE‐selectin had the highest binding efficacy compared to the single antibody coated microspheres. The number of aICAM‐1+aE‐selectin microspheres bound to HUVECs appeared to decrease with increase in wall shear stress in the flow chamber. Moreover, the number of adherent aICAM‐1+aE‐selectin microspheres was significantly higher than the adherent aE‐selectin microspheres under different flow conditions.

Conclusions

We conclude that the adhesion efficiency of the two‐receptor targeting microspheres is significantly higher than the single‐ligand microspheres under various flow conditions and that the drug carrier adhesion ability can be optimized by optimizing the ratio of the two ligands on the surface of the microsphere.
Vascular Endothelial Dysfunction with Age is Related to Increased Sympathetic Nervous System Activity in Women, but Not Men
Authors: Kaplon Rachelle Elizabeth , Walker Ashley Elizabeth , Seals Douglas R
Published: June 2021

We tested the hypothesis that vascular endothelial dysfunction (impaired endothelium‐dependent dilation, EDD) with aging is related to increases in sympathetic nervous system (SNS) activity. In 314 healthy adults aged 18 to 79 years, age was inversely related to EDD (brachial artery flow‐mediated dilation, FMD) (r=−0.30, P<0.001) and positively related to plasma norepinephrine concentrations (PNE) (r=0.49, P<0.001). Brachial FMD was inversely related to PNE in the overall group (r=−0.25, P<0.001), as well as in men only (n=187) and women only (n=127). After controlling for age (multiple regression analysis), brachial FMD remained related to PNE in women (r=−0.24, P<0.01), but not men (r=−0.03, P=0.68). PNE independently predicted FMD in women after further adjustment for conventional risk factors for cardiovascular disease. Indeed, PNE was the strongest independent predictor of FMD in women (r=−0.25, P<0.01). Endothelium‐independent dilation (EID, sublingual nitroglycerin), a measure of vascular smooth muscle sensitivity to nitric oxide, was positively related to PNE in men (P<0.05), but not women (P=0.49). EID did not account for the lack of an independent relation between FMD and PNE in men. These findings suggest that increased SNS activity may play an important role in modulating EDD with aging in women, but not men.

Supported by NIH AG013038, AG015897, AG031617, AG00279, RR025780, AHA 0715735Z
Protease activated receptor‐1 increases phosphorylation of endothelial nitric oxide synthase at site thr495 in human corpus cavernosum endothelial cells
Authors: Mantey Jane Ama , Motley‐Johnson Evangeline D.
Published: June 2021

Endothelial dysfunction, a common symptom of vascular diseases such as erectile dysfunction (ED), encompasses a broad array of physical and molecular defects of the endothelium that are due to low nitric oxide (NO) availability. Protease activated receptor‐1 (PAR1) induces downstream phosphorylation of endothelial nitric oxide synthase (eNOS) at site Threonine‐495 (Thr495) to inhibit NO. We hypothesize that excessive phosphorylation at this site reduces NO availability and contributes to ED, a risk factor for cardiovascular disease. Immortalized human corpus cavernosum endothelial cells (HCCECs) were lysed and PAR1 expression was examined by Western blot analysis. HCCECs were stimulated with either thrombin (10 U/mL) or TFLLR (10μM), the PAR1 activating peptide, at varying time points and eNOS‐Thr495 phosphorylation was determined by Western blot. Results were quantified by densitometry, and demonstrated that PAR1 is expressed in HCCECs. Treatment with thrombin and TFLLR induced maximal downstream phosphorylation of eNOS‐Thr495 at 10 minutes. We believe that this signal transduction pathway is amplified in males with ED, and inhibition of PAR1 will restore NO availability. Our goal is to contribute to the development of novel therapeutic agents that focus on improving NO availability in ED and other cardiovascular diseases. This project was supported, in part, by NIGMS Grant 5SC3GM086336‐02.
Decreased coronary artery development in Wt1 heterozygous mice
Authors: Leung Carmen , Lu Xiangru , Feng Qingping
Published: June 2021

Congenital heart disease is the most common human birth defect which occurs in approximately 1% of all births in Canada. As a major cause of mortality and morbidity, a further understanding of the biological mechanisms underlying congenital heart disease is crucial. Wilms' tumour‐1 (Wt1) was first discovered as a gene mutated in kidney tumour development in children, but has more recently been shown to play an important role in organogenesis. In the developing heart, Wt1 is critical for progenitor cell function. Homozygous Wt1 knockout mice are embryonic lethal with defects in multiple organs, including the heart, kidneys and gonads. Conversely, heterozygous Wt1 (Wt1+/−) mice are reported to be viable and fertile but whether a gene dosage effect exists, specifically related to heart development, remains elusive. In this study, coronary artery formation was investigated in postnatal day one (P1) Wt1+/− mice. Hearts were serially sectioned and immunostained for alpha smooth muscle actin to mark coronary arteries. Amira software was utilized to generate 3‐D reconstructions of the heart to evaluate coronary artery morphology. It was found that coronary artery diameter and coronary artery volume were significantly decreased in Wt1+/−mice compared with Wt1+/+mice at P1. Total body weight, left ventricular, septal and right ventricular myocardial wall thickness were not significantly different between Wt1+/−and Wt1+/+mice at P1. These results demonstrate that expression of both genetic copies of Wt1 is critical for coronary artery development. Ongoing experiments are studying the morphology and function of coronary arteries in the adult Wt1+/− mice. This research is supported by a NSERC Postgraduate Scholarship and the Heart and Stroke Foundation.
Molecular Characterization of Large Conductance, Ca2+‐activated, K+ Channels (BK) in Arteries from Cerebral and Skeletal Muscle Vasculatures
Authors: Nourian Zahra , Li Min , Davis Michael J , Braun Andrew P , Hill Michael A
Published: June 2021

Previous studies have shown functional differences in BK of smooth muscle cells (VSMC) from cerebral and cremaster muscle vasculatures. Specifically, BK from cremaster showed decreased Ca2+ sensitivity resulting in equivalent channel opening at more positive membrane potential compared to cerebral VSMC. Studies were performed to determine if variation in molecular features of BK contributes to differences in activity. The existence of splice variants in the intracellular C terminus domain of the α‐subunit was examined by RT‐PCR. Expression of β1, β2, β3 and β4 subunits was also determined. Total RNA was extracted from isolated arterioles and quantified by absorbance (260/280nm). Equal amounts of RNA were reverse‐transcribed into cDNA and quantitative‐PCR was performed. Two α‐subunit variants, Zero (without splice insert) and STREX‐1 (with a 58 amino acid insert at splice site 2), were identified as identical in cremaster and cerebral arteries by sequencing of RT‐PCR products. Q‐PCR showed the β1 subunit to be the predominant accessory subunit expressed in both vessels. A low level of β4 compared to β1 was observed in both preparations. No evidence was obtained for either β2 or β3 mRNA expression. The results suggest the molecular features of BK are likely similar in the two arteries. Differences in functional properties may relate to subunit stoichiometry of the intact channel or post‐translational mechanisms.
Mechanisms of estrogen's effects on calcium signaling in vascular endothelial cells
Authors: Firkins Rachel , Giles Jennifer , Tran Quang‐Kim
Published: June 2021

17β‐estradiol (E2) exerts many cardioprotective effects via actions on endothelial cells (ECs). It has been observed that E2 increases Ca2+ signals produced in ECs in response to agonists. However, the underlying mechanisms are unclear. Here we have examined the main components of Ca2+ signaling in primary ECs in response to long‐term exposure to physiological concentrations of E2. E2 treatment for 48 hrs did not affect thapsigargin‐induced internal Ca2+ store release or store‐operated Ca2+ entry. However, we observed a 35% reduction in the activity of the calmodulin (CaM)‐dependent plasma membrane Ca2+‐ATPase (PMCA), the main Ca2+ efflux mechanism in ECs. In contrast, calmodulin (CaM) binding to the PMCA is increased by 50%, while total PMCA expression is not affected. Inhibition of pp60src‐mediated tyrosine phosphorylation of PMCA reveals the effects of E2‐induced increase in CaM binding to the PMCA, demonstrating a 30% increase in PMCA activity in E2‐treated cells. Our results suggest that via pp60src‐dependent phosphorylation of PMCA, estrogen decreases Ca2+ efflux, which provides an explanation for the enhanced Ca2+ signals in ECs exposed to E2.
Core Performance Workplace Wellness: Positive impact on cardiovascular disease risk and fitness
Authors: Carlson‐Phillips Amanda , Campbell Bill , Woolf Kathleen
Published: June 2021

Purpose

To compare the effectiveness of two worksite wellness programs.

Methods

Employees participated in a 14‐week cohort‐randomized trial comparing a Traditional Worksite Wellness Program [TWP] and the Core Performance Training and Nutrition Program [CPTNP]. The TWP included a health check, an online health assessment and a meeting with a wellness coach. The CPTNP consisted of the TWP plus a supervised exercise (3 times/wk) and nutrition program (4 meetings). Peak VO2, functional movement, and blood markers of cardiovascular risk were assessed before and after the 14‐wk study. Data were analyzed via a 2x2 repeated measures ANOVA & significance was set at p<0.05.

Results

Participants in the TWP (n=66; 39.5 ± 7.8yrs; 29.2 ± 6.8 kg/m2) and the CPTNP (n=73; 39.1±6.5yrs; 28.6 ± 5.02 kg/m2) were matched for age, gender and activity level. Peak VO2 & functional movement improved by 20% & 5% and 23% & 5% for the CPTNP & TWP programs, respectively. Triglycerides, total &LDL cholesterol concentration decreased 22% & 7%; 5% & 0%; 6% & 0% for the CPTNP & TWP programs, respectively. HDL cholesterol increased by 10% &1% for the CPTNP & TWP programs, respectively. For each variable, a significant interaction was observed demonstrating that the CPTNP had greater improvements compared to the TWP group.

Conclusion

Participants in the CPTNP had significantly better improvements.

Research support from Intel®
Activation of AMP‐activated protein kinase (AMPK) causes vasorelaxation in isolated mesenteric arteries of hypertensive and normotensive rats
Authors: Reid Eric Benjamin , Ford Rebecca J , Durham Kristina K , Rush James W.E.
Published: June 2021

Previous studies have shown that AMPK activation causes relaxation in isolated vessels from healthy animals but this has not been investigated in dysfunctional resistance arteries of hypertensive animals. Here we investigate the effects of AMPK activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) on resistance arteries from normotensive Wistar Kyoto rats (WKY) and Spontaneously Hypertensive rats (SHR). Mesenteric arteries were mounted on a small vessel myograph, pre‐contracted with 10−5M norepinephrine and exposed to increasing concentrations of AICAR (10−6, 10−4, 10−2M). Dose‐dependent relaxation occurred (~10% at 10−6M and ~90% at 10−2M [AICAR], respectively), and did not differ between SHR and WKY. Incubation with the NOS inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 10−4M) blunted relaxation in the WKY at 10−6M AICAR (by ~17% vs. CON, n=6, P<0.01), but did not affect relaxation at higher [AICAR]. Conversely, L‐NAME significantly blunted the relaxation response of SHR rings across all [AICAR] (by ~15–25% vs. CON, n=8, all P<0.01). Thus AICAR causes relaxation in both SHR and WKY mesenteric artery segments, but the relaxation response to AICAR is more NO‐dependent in vessels from SHR compared to WKY. Collectively these findings suggest that AMPK could influence the regulation of resistance vasculature in normotensive and hypertensive animals in an NO‐dependent manner.
High‐resolution spatial quantification of canine coronary collaterals in endo‐ and epicardial layers
Authors: van Horssen Pepijn , van den Wijngaard Jeroen PHM , Siebes Maria , Spaan Jos AE
Published: June 2021

Ischemia and subsequent infarction may occur when blood supply to the heart is impeded by coronary artery disease. Stimulating coronary collateral formation may provide a therapeutic option in patients that cannot be successfully revascularized by conventional surgery. However, detailed information on the mechanism of collateral formation is needed.

In this study an imaging cryomicrotome was used to acquire high resolution data of collateral connections. Coronary arteries in 5 canine hearts were prepared with fluorescent cast material and cut serially at 20μm slices. After each cut, the surface of the remaining material was imaged. Skeletonization of the 3D vasculature allowed defining connectivity and collateral connection identification. On average, 800 collaterals were identified for each heart, diameters ranging from 40 – 200 μm. The number of collaterals was approximately twofold higher in the endocardium compared to the epicardium, even though the measured volume of the endocardium was on average 2.5 times smaller than the epicardial volume. The diameter and length distributions of the collateral segments appeared equal for the two myocardial layers. These characteristics may provide a better understanding of the mechanisms of collateral formation. Supported by the Netherlands Organization for Health Research and Development (ZonMw 91105008and 91611171), and by FP7‐ICT 2007224495 (euHeart).
Mineralocorticoid receptor blockade does not affect arterial compliance in middle‐aged and older adults with and without metabolic syndrome
Authors: Yoo Jeung‐Ki , Hwang Moon‐Hyon , Luttrell Meredith J. , Cernosek Molly M. , Meade Thomas H. , English Mark W. , Christou Demetra D.
Published: June 2021

Aging and metabolic syndrome (MS) are associated with decreased arterial compliance but the underlying mechanisms are not completely understood. Aldosterone and activation of the mineralocorticoid receptors (MR) are thought to be elevated in MS and may contribute to reduced arterial compliance. We hypothesized that chronically elevated activation of MR is partly responsible for reduced arterial compliance in aging/MS and that MR blockade would lead to greater improvements in carotid artery compliance (CAC: high resolution ultrasonography and applanation tonometry) in middle‐aged (MA) and older (O) adults with vs. without MS. To test this hypothesis, we administered 100 mg of Eplerenone (MR blocker) or placebo per day for 4 weeks in a randomized, double blind, crossover design, in 6 MA/O adults with and 14 without MS. At baseline, brachial blood pressure (BP) was higher (mean BP: 98±2 vs. 90±3 mmHg, P=0.03) and CAC was lower (0.12±0.02 vs. 0.19±0.03 mm2/mmHg, P=0.03) in the MS group. MR blockade reduced brachial BP similarly in both groups (delta mean BP: −6±2 vs. −3±1 mmHg, P=0.1, with vs. without MS) but it did not affect CAC in either group (delta CAC: −0.01±0.01 vs. −0.02±0.03 mm2/mmHg, P=0.4, with vs. without MS). In conclusion, arterial compliance in MA/O adults with or without MS is not affected by short‐term MR blockade.

Supported by AHA Grant 0865117F and NIH Grant AG032067.
P311‐null mice are hypotensive due to a systemic decrease in TGF‐beta level and activity
Authors: Badri Kameswara Rao , Aramgam Sree Latha , Dhru Urmil , Cao Jun , Schuger Lucia
Published: June 2021

P311 is an 8 kDa protein highly expressed in brain and vascular smooth muscle (VSM). Telemetry and VPR tail cuff plethysmography demonstrated that p311‐null (P311 KO) mice exhibit pronounced systemic hypotension in the absence of phenotypic abnormalities. Myography and collagen gel contraction studies revealed a severe decrease in blood vessel and VSM cell contraction respectively. The hypofunctionality of the VSM contractile apparatus was confirmed by immunoblot analysis. Since we and others found that P311 is involved in TGF‐β regulation and since TGF‐β participates in blood pressure control, we determined the level and activity of TGF‐β in the VSM and blood of P311 KO mice. These studies revealed a significant downregulation in all TGF‐β1‐3 levels and activity. Treatment with exogenous recombinant TGF‐β1 or 3, but not TGF‐β2 partially rescued the defective contractile phenotype, whereas combined treatment restored normal contractility in vivo and in vitro. Our studies demonstrated that P311 controls the systemic levels and activity of TGF‐β1‐3 and thereby plays a critical role in vascular homeostasis. This work is supported by NHLBI.
TANNIC ACID AN INHIBITOR FOR ANGIOTENSIN TYPE 1 RECEPTOR AND HYPERTENSION IN SPONTANEOUSLY HYPERTENSIVE RATS
Authors: Gumaste Upendra , Snyder Russell , Koganti Sivaramakrishna , Karamyan Vardan , Thekkumkara Thomas
Published: June 2021

Our study in rat liver cells showed that tannic acid (TA), a polyphenol present in fruits and vegetables down‐regulates angiotensin type 1 receptor (AT1R) expression. However, in vivo effects of TA on blood pressure and AT1R expression were not known. In this study we examined the possible protective effects of TA on blood pressure, AT1R receptor protein and mRNA expression in target tissues of spontaneously hypertensive rats (SHR). Animals with a base line mean arterial pressure (MAP); 142.404 ± 9.303 mmHg were daily treated (IP) with TA (5mg/kg body weight). Treated animals showed reduced MAP (114.111 ± 2.751 mmHg) over a period of 4 weeks compared to animals treated with vehicle control (141.067 ± 7.642 mmHg). At the end of the study, withdrawal of TA treatment for a week reversed its inhibitory effects (128.344 ± 9.133 mmHg). Consistent with the reduction in blood pressure [3H]Angiotensin II specific binding studies on membrane preparations showed significant reduction in AT1R receptor expression in heart and kidney with no change in the liver. However, real‐time RT‐PCR analysis showed significant reduction in AT1R mRNA expression in the liver. Furthermore, we observed AT1R blockade‐induced weight loss (9–10%) in these animals, which is consistent with the literature. These results suggest for the first time that long‐term therapy with TA can prevent hypertensive disorders in SHR. Supported by NIH Grant DK072140
The Hsp90‐dependent and Indepedent roles of FKBP38 in the biogenesis of CFTR
Authors: Banasavadi‐Siddegowda Yeshavanth Kumar , Mai Junbo , Wang Xiaodong
Published: June 2021

The cellular chaperone machinery regulates the biogenesis of nascent ion channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). FK506‐binding protein 38 (FKBP38), an immunophilin anchored to the endoplasmic reticulum (ER) membrane, was identified as a component of a chaperone network associated with CFTR in the ER. Like FKBP52, FKBP38 binds Hsp90 through its tetratricopeptide repeat (TPR) domain. FKBP52 serves as an Hsp90 cochaperone in the activation of steroid receptors. However, the functional relationship between FKBP38 and Hsp90 in the context of CFTR biogenesis remains unclear. To understand the regulation of CFTR biogenesis by FKBP38, we introduced a series of domain‐specific, shRNA‐refractory mutants of FKBP38 into an FKBP38‐deficient cell line. Our data support a central role for the peptidylprolyl cis/trans isomerase in FKBP38‐mediated folding of CFTR and a moderate regulatory role for Hsp90 in FKBP38‐mediated folding and/or quality control of CFTR in the ER.
Optimization of in vitro translation using different human cell lines
Authors: Dhodda Vinay K , Jensen Penny , Deshpande Priya , Mcbride Aaron , Kilmer Gregory , Hommema Eric , Choi Jae , Webb Brian , Vattem Krishna M.
Published: June 2021

Culturing of mammalian cells for the purpose of protein expression is a time consuming and expensive process. An effective alternative to cell culture is cell‐free protein expression (i.e., in vitro translation) using extracts prepared from mammalian cells. We have used several immortalized human cell lines, including HeLa and HepG2, to make translation competent extracts ideal for expressing proteins. We have also optimized the HeLa in vitro translation system for higher protein expression. To highlight the flexibility of our translation system, we have demonstrated expression of 5 different proteins in a single reaction. We have also shown the compatibility of our system with high throughput protein expression using a PCR‐based approach. These results indicate the utility of the HeLa in vitro translation system for expression of functional human proteins.
Ca2+‐activated Cl− channels contribute to regulating store‐ and receptor‐operated Ca2+ entry in human pulmonary arterial smooth muscle cells
Authors: Yamamura Aya , Yamamura Hisao , Yuan Jason X.‐J.
Published: June 2021

Cytosolic Ca2+ ([Ca2+]cyt) plays a fundamental role in regulating cell functions in pulmonary arterial smooth muscle cells (PASMC). A rise in [Ca2+]cyt triggers pulmonary vasoconstriction and stimulates PASMC proliferation. [Ca2+]cyt is mainly increased by Ca2+ release from intracellular stores and Ca2+ influx through plasmalemmal Ca2+‐permeable channels. Given the high concentration of intracellular Cl− in PASMC, Ca2+‐activated Cl− (ClCa) channels play an important role in regulating membrane potential and cell excitability of PASMC. In this study, we examined whether ClCa channels were involved in regulating [Ca2+]cyt in PASMC via store‐operated (SOCE) and receptor‐operated (ROCE) Ca2+ entry. [Ca2+]cyt was measured in human PASMCs loaded with fura‐2/AM using a Nikon digital fluorescence imaging system. SOCE was induced by passive depletion of Ca2+ from the sarcoplasmic reticulum (SR) by cyclopiazonic acid (10 μM). Inhibition of ClCa channels by 100 μM niflumic acid significantly reduced both transient and plateau phases of SOCE. In addition, angiotensin II‐mediated increase in [Ca2+]cyt via ROCE was markedly attenuated by the ClCa channel inhibitors, niflumic acid, flufenamic acid or DIDS. These results suggest that function of ClCa channels contributes to regulating [Ca2+]cyt by affecting SOCE and ROCE in human PASMC. Research Funding Source: NIH grants (HL066012 and HL 098053).
Expression of TASK‐2 and its upregulation by B cell receptor stimulation in WEHI‐231 mouse immature B cells
Authors: Shin Dong Hoon
Published: June 2021

Stimulation of B cell receptors (BCR‐ligation) induces apoptosis of immature B cells, which is critical to the elimination of self‐reactive clones. In the mouse immature B cell line WEHI‐231, we previously reported two types of background K+ channels with large‐ (~ 300 pS, LKbg) and medium (~ 100 pS, MKbg) conductance in divalent cation‐free conditions. LKbg has been recently identified as TREK‐2, however, the molecular nature of MKbg is not known yet. In the present study, we found that the background K+ conductance of WEHI‐231 was markedly increased by BCR‐ligation. A single channel study revealed that MKbg activity was increased by BCR‐ligation, and that the biophysical properties of MKbg were consistent with those of TASK‐2. The expression of TASK‐2 and its upregulation by BCR‐ligation was confirmed by RT‐PCR and immunoblot assays in WEHI‐231. The BCR‐ligation‐induced increase of K+ current was prevented by calcineurin inhibitors (cyclosporine A or FK506), and also by TASK‐2 specific siRNA transfection (si‐TASK‐2). Furthermore, si‐TASK‐2 attenuated the apoptosis of WEHI‐231 caused by BCR‐ligation. TASK‐2 activity and its mRNA were also confirmed in the primary splenic B cells of mouse. Summarizing, the authors report for the first time the expression of TASK‐2 in B cells, and surmise that the upregulation of TASK‐2 by BCR‐ligation is associated with the apoptosis of immature B cells.
ATP stimulation of volume regulation in cultured human astrocytoma cells
Authors: Woods Megan , Olson James
Published: June 2021

Introduction

Understanding cell volume control of brain cells will lead to better treatments for patients suffering from brain edema following trauma and other conditions. We examined the role P2Y receptors play in cell volume regulation using cultured 1321N1 human astrocytoma cells.

Methods

Cells were removed from the growth surface and suspended in isoosmotic phosphate buffered saline (Iso‐PBS) containing (in mM): NaCl (147), KH2PO4 (0.5), Na2HPO4 (3.2), CaCl2 (1), MgCl2 (0.5), dextrose (5), and KCl (2.7) (pH=7.3, osmolality=290 mOsm) at 37 °C for 30 min. Cell volumes were determined using the Coulter method at various times after cells were resuspended in Iso‐PBS or similar solutions with osmolalities altered by changing concentrations of NaCl.

Results

We observed a linear relationship of initial volume versus (osm)−1 for cells in osmolalities ranging from 150 mOsm to 600 mOsm with an intercept at 45.3±1.3% of the volume measured in Iso‐PBS. Maximal cell swelling in 150 mOsm PBS occurred 3–5 min after suspension. After 10 min and 30 min in this hypoosmotic condition, cell volume had recovered by 18.3±5.1% and 48.4±2.2%, respectively. When 100 μM ATP was added to the 150 mOsm PBS solution, the rate of volume recovery was significantly faster.

Conclusion

132N1 cells demonstrate regulatory volume decrease in hypoosmotic conditions which may be stimulated by purinergic receptors. Supported by NIH R25GM086257.