bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinoic acid application transiently increases microglia and macrophages after optic nerve injury in frog Rana pipiens
Authors: De La Rosa-Reyes Valeria , Duprey-Díaz Mildred Vanessa , Blagburn Jonathan M , Blanco Rosa Esther
Published: April 2020

Retinoic acid (RA) plays major roles during nervous system development, and during regeneration of the adult nervous system. We have previously shown that RA signaling pathway components are upregulated after optic nerve injury and that exogenous application of RA greatly increases the survival of axotomized retinal ganglion cells (RGCs). We have recently reported that the application of growth factors increases the number of macrophages in the regenerating optic nerve. The objective of the present study is to study in more the effects of RA application on the macrophages and microglia in the optic nerve after injury. We performed optic nerve crush and applied to the nerve either saline solution, retinoic acid or clodronate liposomes. We examined the optic nerves at one week, and two weeks, after axotomy with immunocytochemistry and electron microscopy. Our results show that application of RA to the optic nerve causes a significant increase in the number of macrophages and microglia one week after optic nerve injury. The macrophages present have considerable phagocytic activity and are consistently labeled with M2‐type macrophage markers. Numbers of activated microglia also increase near the injury site. Two weeks after axotomy and RA treatment there are fewer macrophages, and their ultrastructure shows less phagocytic activity. Clodronate liposome treatment at the time of axotomy apparently eliminates the macrophages present in the nerve at one week. We measured RGC survival at six weeks after axotomy and clodronate treatment and found no obvious effect of this treatment. In conclusion, the application of retinoic acid increases the number of macrophages and microglia present in the nerve after optic nerve injury. Further studies will explore if this effect plays a role in the success of optic nerve regeneration.

Support or Funding Information

REB is supported by NIH grant GM116692, JMB by NIH grant NS081726 and VDR by NIGMS‐RISE R25 GM061838
Studies of specific yeast prion chimera to test hypotheses about prion propagation and Hsp104‐mediated prion elimination
Authors: MacElroy Andrew S. , Schock Sydney F. , Singh Atul K.
Published: April 2020

In Saccharomyces cerevisiae, at least 10 proteins are known to form amyloid‐based prions which propagate in cell populations due to continuous fragmentation by molecular chaperones. Hsp104, Hsp70, and the Hsp40 Sis1 are all universally essential to this process; however, prions differ in their specific requirement for Sis1 activities, and some require additional Hsp40 chaperones. For reasons which are still being explored, only one prion, [PSI+], is eliminated when Hsp104 is overexpressed. We are utilizing prion‐chimeras originally developed by the Lindquist lab at MIT to test various hypotheses regarding prion propagation and Hsp104‐mediated elimination, including the role of amino acid content of the prion‐forming domain in determining prion‐Hsp40 requirements. Additionally, our investigations explore the hypothesis that the specificity for Hsp104‐mediated prion elimination is due to a unique protein region of the [PSI+]‐forming protein Sup35 known only as the “M” domain. Preliminary results indicate that the presence of the M domain is an important, if not essential, component in determining the ability of a prion to be eliminated by Hsp104 overexpression.

Support or Funding Information

This work was supported by the Lafayette College Chemistry Department, the EXCEL research scholarship program (
www.lafayette.edu), the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606, and the Henry Dreyfus Teacher Scholar Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Dreyfus Foundation.
Functional Platelet‐derived Mitochondria Induce the Release of Neutrophil Microparticles
Authors: Boudreau Luc H. , Foulem Robert D. , Léger Jacob L.
Published: April 2020

INTRODUCTION

Inflammation is an essential process of the host defense against infections, illness, or tissue injuries. However, unregulated inflammation has been associated with chronic inflammatory auto‐immune diseases, such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. Polymorphonuclear neutrophils (PMNL) are amongst the immune cells involved in the acute inflammatory response used to fight bacterial infections. Once activated, PMNL release inflammatory mediators, enzymes and large quantities of microparticles in the extracellular milieu to recruit various immune cells required to fight the invading pathogens. Recent evidence also shows that platelets (PLTs), well known for their coagulation proprieties, are also implicated in the body’s inflammatory response. Interestingly, activated PLTs can release fully functional mitochondria in the extracellular milieu. Known as the powerhouse of the cell, the mitochondria share similar characteristics with bacteria. Therefore, we hypothesize that PLTs‐derived mitochondria present in the extracellular milieu, acting in a similar way as bacteria, induce a sterile inflammatory response that involves the PMNL.

OBJECTIVES

The main objective of this study is to investigate the sterile inflammatory response of PMNL caused by the exposure of PLTs‐derived extracellular mitochondria.

METHODS

Blood was obtained from healthy consenting donors, then PMNL and PLTs‐derived mitochondria were isolated and purified. Following the co‐incubation of PMNL with various physiological doses of PLTs‐derived mitochondria, a characterization of the interaction between PMNL and PLTs‐derived mitochondria and an investigation of the inflammatory proprieties of PMNL was performed using flow cytometry, confocal microscopy, and high‐resolution respirometry.

RESULTS

Our data demonstrates that PLTs‐derived mitochondria associate with PMNL as quickly as 2 hours. This results in an increase of the PMNL intracellular calcium content and a 2.47‐fold mitochondrial‐dependent increase in oxygen consumption. Data also shows that PLTs‐derived mitochondria significantly induce, in a dose‐dependent manner, the release of PMNL microparticles.

CONCLUSIONS

This study provides a new insight into the role of PLTs‐derived mitochondria in the context of sterile inflammation. Specifically, the characterization of its interaction with PMNL reveals a novel modulator in the fundamental inflammatory response. The knowledge gained from this study provides insight into the mechanism of sterile inflammation in auto‐immune diseases.

Support or Funding Information

Canadian Institutes of Health Research, New Brunswick Health Research Foundation, New Brunswick Innovation Foundation
Rapamycin does not reproduce the alterations to Caco‐2 transepithelial electrical resistance caused by the knockdown of Raptor
Authors: Kaur Harleen , Moreau Regis
Published: April 2020

Intestinal homeostasis is achieved through well‐coordinated interactions between the mucus layer, the epithelial layer and the immune cells of the lamina propria. Owing to its proximity with the luminal content, the intestinal epithelium is exposed to insults of chemical, physical or dysbiotic nature. These insults may perturb the epithelium permeability and trigger a localized immune response, which if not controlled may progress to damage the epithelium. Although the mechanistic target of rapamycin complex 1 (mTORC1) is deactivated in differentiated enterocytes, mTORC1 becomes active under repeated insult or injury to the epithelium and participates in epithelial cell repair. However, many aspects of mTORC1 re‐activation remain to be elucidated. To investigate the effects of graded levels of mTORC1 activity on epithelial barrier function, we knocked down (>90% down) mTORC1 signaling‐associated proteins, Raptor and TSC2, to create three Caco‐2 cell lines in which, after full differentiation in Transwell®, mTORC1 activity was either normal (shScramble cells), low (shRaptor cells, mTORC1 activity 50% lower than shScramble cells) or high (shTSC2 cells, mTORC1 activity 80% higher than shScramble cells). When compared with shScramble cells, shRaptor cells had higher abundance of p‐(Thr172)AMPK (+46%, P<0.05), lower abundance of p‐(Ser757)ULK‐1 (−50%, P>0.05), higher autophagic flux (+180%, P<0.05), and lower expression of pore‐forming tight junction protein Claudin‐2 (−65%, P<0.05). In contrast, shTSC2 cells had higher levels of p‐(Ser757)ULK‐1 (+100%, P<0.05) and Claudin‐2 (+250%, P<0.05) compared with shScramble cells. These mTORC1‐related changes were accompanied by significant alteration to the Caco‐2 transepithelial electrical resistance (TEER), which was 180% higher in shRaptor cells vs. shScramble cells, and conversely 60% lower in shTSC2 cells vs. shScramble cells. Although rapamycin suppressed mTORC1 activity in these cells, rapamycin caused a modest 34% (P<0.05) increase of TEER in shScramble cells, suggesting that factors other than mTORC1 ablation were responsible for the high TEER of shRaptor cells. The use of chloroquine (an inhibitor of autophagosome‐lysosome fusion) showed that autophagy alone could not explain the strong electrical resistance of shRaptor cells. We are currently investigating whether TSC2, whose expression increases as Caco‐2 cells differentiate concomitantly with mTORC1 silencing, might regulate paracellular permeability and tight junction assembly in the epithelium.

Support or Funding Information

USDA‐NIFA
Unraveling the metabolic and machinery constraints on protein secretion through a novel systems biology framework
Authors: Lewis Nathan E.
Published: April 2020

In mammalian cells, metabolism is a core process driving homeostasis, but variations in other cell processes largely define cell type identity and cell‐type specific functions. The profile of secreted and membrane proteins show substantial cell‐type specificity and drive many tissue specific functions. These proteins, encoded by up to 1/3 of mammalian protein‐coding genes, include hormones, membrane proteins, and extracellular enzymes, and these are synthesized and trafficked through the secretory pathway. The pathway complexity, however, obfuscates its impact on the secretion of different proteins. Unraveling its impact on diverse proteins is particularly important since the pathway is implicated in many diseases and harnessed for biopharmaceutical production. Through the use of network reconstruction approaches and protein interaction assays, we have mapped out the core secretory pathway and integrated it with our genome‐scale metabolic models of human, mouse, and Chinese hamster ovary cells. We first deploy graph‐based approaches to evaluate the dependency of protein secretion on diverse human secreted proteins. We then deployed constraint‐based modeling to quantify the bioenergetic demands for the synthesis and secretion of these proteins. Finally, we deployed these models to engineer mammalian cells for enhanced secretion of high‐value biologic drugs. Thus, we present a platform that enables the study and engineering of the mammalian secretory pathway and metabolism for systems biotechnology.

Support or Funding Information

NIGMS R35 GM119850 and the Novo Nordisk Foundation Center for Biosustainability NNF10CC1016517
Comparison of Association Between Physical Activity and Resting Metabolic Rate on Korean Young and Middle‐Aged Adults
Authors: HWANG HYEJUNG , Jung Wonsang , Kim Jisu , Park Hun-young , Lim Kiwon
Published: April 2020

Purpose

The purpose of this study was to investigate the correlation of body composition, RMR, and PA between Korean young and middle‐aged adults.

Methods

The total subjects were fifty‐three [male n=23, female n=30], thirty‐four healthy young adults [male n=18, female n=16] and nineteen middle‐aged adults [male n=5, female n=14]. All participants measured body composition and Resting metabolic rate (RMR) after overnight fasting (≥8h). The investigation of physical activity was using a Korean version of the WHO Global Physical Activity Questionnaire (GPAQ).

Results

Body composition did not present a significant difference between young adults and middle‐aged adults. The whole‐body bone mineral density (BMD) and bone mineral contents (BMC) was significantly lower in middle‐aged adults. Total blood cholesterol (TC) and blood glucose were significantly higher in middle‐aged adults (TC; 195.21 ± 43.34, Glucose; 103.57 ± 12.61 mg/dL). The RMR was significantly lower in middle‐aged adults (1619.57 ± 290.28 kcal/day) than young adults (1894.37 ± 405.00 kcal/day). In the middle‐aged adults physical activity (PA). PA (METs, min, EE) has inverse correlations with fat mass (FM, kg and %), and blood TG level in young adults. The middle‐aged adults indicate that PA has a significant positive correlation with lean body mass (LBM), FM (%), and RMR. Furthermore, PA EE also shows significant interrelatedness with BMC among the middle‐aged adults.

Conclusion

These results demonstrate high levels of PA preserve LBM and RMR in middle‐aged adults, furthermore, young adults demanded more PA to induce change in body composition.

Support or Funding Information

This work
is supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF‐2019S1A5B8099542).
Role of Apontic in ASP Development in Drosophila melanogaster
Authors: Lozano Camila , Srivastava Ajay
Published: April 2020

The air sac primordium (ASP), found in the Wing Imaginal Disc of Drosophila melanogaster, can be examined as a model for invasive behavior due to the properties it inhibits during development which imitate those necessary for tumor progression. The Janus kinase/signal transduction and activation (JAK‐STAT) signaling pathway plays a considerable role during the development of the ASP and also has its own implications regarding tumor progression and metastasis. The gene apontic (apt), which codes for a protein expressed in the ASP, is known to be a negative regulator of JAK‐STAT. In this study we investigated whether downregulation of the gene apontic had an effect on the invasive behavior of the ASP through the disruption of JAK‐STAT. Using the GAL4/UAS system for targeted gene expression, we were able to knock down apontic and compare ASP development in affected lines vs. control lines. Through the downregulation of apontic, we observed an increased number of under‐developed ASPs. The data presented is able to provide further understanding into the factors that could potentially play a role in the invasive behavior of tumors.

Support or Funding Information

Dr. Ajay Srivastava, Aref Ranjbar, WKU Biotechnology Center, KBRIN AREA and IDeA Grants funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health Grant# P20GM103436, Gatton Academy Research Internship Grant (RIG)
Deletion of SIRT1 in the skeletal muscle decreases type IIa oxidative muscle fiber in mice.
Authors: Hosoda Ryusuke , Kuno Atsushi , Asakura Seidai , Horio Yoshiyuki
Published: April 2020

Background and Aim

SIRT1 is an NAD+‐dependent protein deacetylase, which exerts cytoprotective effects. We previously reported that resveratrol, an activator of SIRT1, improves exercise tolerance with upregulation oxidative type of muscle fibers in a mouse model of Duchenne muscular dystrophy. SIRT1 deacetylates and activates PGC‐1α, a co‐activator to promote expression of oxidative muscle fibers. The purpose of this study was to examine whether SIRT1 maintains oxidative muscle fibers for exercise capacity in the skeletal muscle using muscle‐specific SIRT1 knockout mice (SIRT1MKO).

Methods and Results

We first compared the expression level of SIRT1 in several types of skeletal muscles in wild‐type mouse (WT). The SIRT1 protein level was the highest in the soleus muscle (+3.5‐fold), which is mainly made up of oxidative fibers, compared to the other glycolytic muscles such as quadriceps, gastrocnemius, tibial anterior, and extensor digitorum longus muscles. The SIRT1 mRNA level was most abundant also in the soleus. Immunohistological analysis using soleus muscle sections showed that the percentage of type IIa, one of oxidative muscle fibers, was significantly lower in the SIRT1MKO than that in WT (42% vs. 56%) at 26–32 months old. In contrast, the percentage of glycolytic type IIx+IIb muscle fibers was higher in the SIRT1MKO (15%) compared to WT (11%). Treadmill running distance at 30 months old was significantly shorter in SIRT1MKO (78±8 m) than that in WT (592±10 m).

Conclusion

These results suggest that deletion of SIRT1 in the skeletal muscle reduces exercise capacity by decreases type IIa oxidative muscle fiber.
Training effects on central and peripheral components of force, in old healthy subjects: the role of central command.
Authors: Venturelli Massimo , Targhetta Gino , Schena Federico
Published: April 2020

Aim

The aim of this study, was to investigate the role of central command (CC), during resistance training in old healthy men. For this purpose, maximal voluntary contraction (MVC), percentage of neuromuscular activation (%VMA) and rest twitch (RT) were determined before and after 4 weeks of quadriceps electro‐stimulation (ES), or voluntary isometric training (VT).

Methods

12 old (65–70 years) and 12 young male subjects (20–25 years) were recruited for this study. ES was performed by a commercially available electrical stimulator. ES included 15 minutes of isometric‐evoked contractions (6 s contraction/ 11 s of recovery) in the quadriceps, 3 times a week for a total of 12 training sessions. VT was performed after a washout period of 8 weeks, matching the force‐output recorded during the ES.

Results

MVC was minimally increased (+4%) after ES in old subjects, while young subjects showed a larger amelioration (+28%). After ES, %VMA was not changed (+3%) but RT was increased (+16%) in old individuals, while in the young subjects the changes were greater, +17% and +78%, respectively. Both, young and old subjects exhibited after VT significant increases in MVC +16% and +42% respectively. %VMA and RT in old subjects were increased by +8 and +26%, respectively. Similarly, %VMA and RT were increased in young individuals (+9% and +38%).

Conclusions

The results of the current study, suggest that the CC has a primary role to determine improvement of MVC, especially in old subjects. In fact, after the training period, the greater force ameliorations were retrieved in the consequences of VT. These changes were coupled with increases in central (%MVC) and peripheral (RT) components of force. While, the lack of improvements in %VMA after ES in old subjects suggest that the force amelioration was primarily due to peripheral factors.

Support or Funding Information

This project was supported by the Department of Excellence Grant ‐ Molecular and functional basis of successful aging and frailty SAFe (Successful Aging Frailty) ‐
Exploring How Compartment‐specific Changes in NAD Biosynthesis Influence the Response to Endurance Training
Authors: Lakeland Thomas Victor , Samsudeen Azrah , Metcalfe Louise , Govindaraju Hemna , Turner Nigel
Published: April 2020

Nicotinamide adenine dinucleotide (NAD) plays a vital role in the maintenance of health, and is known to decline with ageing and with metabolic and chronic disease states. Restoration of declining NAD levels—which can be achieved through diet, exercise, and pharmaceutical‐based interventions—has been demonstrated in various organisms to associate with increased longevity and a return to healthy physical function. Our group has previously observed alterations in metabolic processes and in muscle morphology in mice overexpressing the NAD biosynthetic enzyme nicotinamide mononucleotide adenylyl transferase (NMNAT) to target nuclear (NMNAT1) and mitochondrial (NMNAT3) NAD upregulation. In the present investigation, both NMNAT1 and NMNAT3 mice and their respective wildtype littermates were exposed to a six‐week progressive endurance training programme. Endurance capacity, oral glucose tolerance (oGTT), various measures of circulating and tissue lipid and glucose content, and skeletal muscle protein expression were assessed. Despite substantially reduced muscle mass in the NMNAT1Tg/+ group and lower overall performance in the NMNAT3Tg/+ group, the ability to adapt to exercise training was not diminished in either group (improvement above baseline of 147.7 ± 40.1% and 136.6 ± 28.6% for NMNAT1 TG and WT, respectively; and 98.4 ± 36.1% and 72.3 ± 20.5% for NMNAT3 TG and WT, respectively; n = 8–9). Both training and NMNAT1 overexpression were correlated with reduced glucose levels during oGTT, with signs of improved insulin sensitivity evident in the NMNAT1Tg/+ groups; no evidence of improved glucose handling was evident with NMNAT3 overexpression. NMNAT1—but not NMNAT3—overexpression was associated with elevated glycogen storage, with no discernible impact on lipid handling. NMNAT3 overexpression was associated with reduced serum and liver TAG levels in the sedentary but not the trained group. Our results suggest that, despite alterations in glucose and lipid handling, overexpression of NMNAT in the nucleus or the mitochondria does not result in a reduced capacity for adaptation to endurance training. NMNAT overexpression within the nucleus appears to positively impact glucose metabolism; whereas overexpression within the mitochondria appears to alter lipid metabolism in sedentary animals, and these differences diminish with exercise training.

Support or Funding Information

NHMRC APP1128351
High Sodium Diet – Role in Renal Sympathodepression
Authors: Rodionova Kristina , Ditting Tilmann , Fahrmeier Franziska , Ott Christian , Schmieder Roland , Schiffer Mario , Amann Kerstin , Veelken Roland Albert
Published: April 2020

Afferent renal nerve pathways are likely involved in the development of salt sensitive hypertension. We recently reported that intrarenal NaCl elicited a long‐lasting sympatho‐depression via a neuro‐humoral TRPV1 dependent and tachykinin mediated renal afferent nerve pathway. We now wanted to test the hypothesis that high sodium intake impairs this afferent sympatho‐depressory mechanism.

Respective groups were put on tap water, 0.9 % saline for drinking or chow containing 8% NaCl. Cultured dorsal root ganglion neurons (DRG Th11‐L2) of rats with renal afferents were investigated in current clamp mode to assess action potential generation during current injection. Rats were equipped with femoral catheters for blood pressure (BP) & heart rate (HR) assessment, drug application, a renal arterial catheter for intrarenal administration (IRA) of NaCl boil (10 % NaCl, 10 μl) or Capsaicin (CAP 3.3, 6.6, 10, 33*10‐7 M, 10 μl) and a bipolar electrode for renal sympathetic nerve activity (RSNA) recordings; eventually an intravenous (iv) bolus of the NK1‐receptor blocker RP67580 (10*10‐3M, 15 μl) was administered. Results are mean±SEM.

In neurons from rats on high salt diet, but not on 0.9 % saline or tap water, the relation of tonic highly active neurons to less active neurons shifted towards less active units (62% tonic neurons in a control group vs. 40% on high salt diet and 63% on saline, significant, p<0.05, z‐test, mean +/−SEM). However, cultured renal neurons from rats on 0.9% saline or on high salt diet exhibited increased action potential production upon stimulation (Controls 13,3+/11,03 APs/600ms vs. saline 19,8+/−2,33 APs/600ms vs. high salt diet 22,2+/−4,54 APs/600ms, significant, <0.05, t−test, mean+/−SEM). 10% NaCl boli IRA induced decreases of RSNA from baseline 4.1±0.6 μV*sec to 2.2±0.8 μV*sec (10% NaCl, p<0.05) impaired in rats on 8% NaCl chow. (Suppressed RSNA unmasked in all groups by an i.v. NK1‐inhibitor).

In rats on high salt diet highly active tonic neurons with renal afferents in vitro decreased at the expense of less active phasic neurons in spite of tonic neurons producing more action potentials upon stimulation. The sodium inducible long‐lasting sympatho‐depression via a neuro‐humoral tachykinin mediated afferent renal nerve pathway got eventually impaired.
Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening and Hypertension
Authors: Wu Jing , Fang Shi , Hu Chunyan , Lu Ko-Ting , Sigmund Curt D.
Published: April 2020

Mutations in CULLIN3 gene (causing in‐frame deletion of exon 9) cause human hypertension (HT), which is likely to be driven by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 mutant causes vascular dysfunction and HT via augmented RhoA/Rho‐kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo, we bred the conditionally activatable Cul3Δ9 mice with tamoxifen‐inducible Tie2‐CREERT2 mice. The resultant mice (E‐Cul3Δ9) developed nocturnal hypertension (Night time peak systolic BP, E‐Cul3Δ9: 138±3 vs Control: 121±4 mmHg, p<0.01) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, p<0.01). No difference was seen in daytime BP. Nitric oxide synthase (NOS) inhibitor L‐NAME induced smaller increases in nocturnal peak SBP and DBP in E‐Cul3Δ9 mice (15±1 vs 27±3 mmHg, p<0.01), suggesting their NOS activity is low. Of note, E‐Cul3Δ9 mice exhibited impaired endothelial‐dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%, p<0.05) and cerebral resistance basilar artery (41% vs 77%, p<0.01). No difference in smooth muscle function was observed. To determine the molecular mechanisms, we isolated primary aortic endothelial cells from mice carrying the inducible Cul3Δ9 construct and induced Cul3Δ9 expression in vitro using adenovirus carrying Cre recombinase gene. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9‐induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin3/PP2A/phospho‐eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin‐3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.

Support or Funding Information

NIH and AHA
Caloric restriction reverses left ventricular hypertrophy through the regulation of cardiac iron homeostasis in ob/ob mice
Authors: An Hyeong Seok , Roh Gu Seob
Published: April 2020

Leptin‐deficient mice manifest obesity, insulin resistance, and left ventricular hypertrophy (LVH); however, LVH’s mechanisms are not fully understood. Cardiac iron dysregulation has been recently implicated in cardiomyopathy. Here we investigated the protective effects of caloric restriction on cardiac remodeling in impaired leptin signaling obese mice. RNA‐seq analysis was performed to assess the differential gene expressions in the heart of wild‐type and ob/ob mice. In particular, to investigate the roles of caloric restriction on iron homeostasis‐related gene expressions, 10‐week‐old ob/ob mice were assigned to ad libitum or calorie‐restricted diets for 12 weeks. Male ob/ob mice exhibited LVH, cardiac inflammation, and oxidative stress. Using RNA‐seq analysis, we identified that an iron uptake‐associated gene, transferrin receptor, was upregulated in obese ob/ob mice with LVH. Caloric restriction attenuated myocyte hypertrophy, cardiac inflammation, fibrosis, and oxidative stress in ob/ob mice. Furthermore, we found that caloric restriction reversed iron homeostasis‐related lipocalin 2, divalent metal transporter 1, transferrin receptor, ferritin, ferroportin, and hepcidin expressions in the heart of ob/ob mice. These findings demonstrate that the cardioprotective effects of caloric restriction result from the cellular regulation of iron homeostasis, thereby decreasing oxidative stress, inflammation, and cardiac remodeling. We suggest that decreasing iron‐mediated oxidative stress and inflammation offers new therapeutic approaches for obesity‐induced cardiomyopathy.

Support or Funding Information

This work was supported by a grant of the Basic Science Research Program through the National Research Foundation of Korea (2015R1A5A2008833).
The Role of Hyperpolarization‐Activated Cyclic Nucleotide‐Gated Channels in Ileus Development
Authors: Gergely Bence , Sipos Ádám , Docsa Tibor , Uray Karen
Published: April 2020

Introduction

Ileus, a decrease or cessation of intestinal contractile activity, affects millions of trauma, post‐operative, and critically ill patients, resulting in longer hospital and ICU stays and increased patient complications. Mechanosensitive ion channels play a role in both the physiological and pathological responses to mechanical stimuli. We found that hyperpolarization‐activated cyclic nucleotide‐gated channel 2 (HCN2) was down‐regulated in edematous intestinal smooth muscle layers. However, the effects of altered HCN channel activity on intestinal contractile activity are largely unknown.

Objective

The purpose of the study was to investigate the effects of HCN inhibition on intestinal contractile activity. We hypothesize that inhibition of HCN will decrease intestinal contractile activity. Furthermore, we hypothesize that HCN activity is inhibited by mechanical stretch and/or inflammatory mediator changes that occur during the development of ileus.

Methods

The effects of HCN inhibitors on intestinal contractile activity were investigated ex vivo in an organ bath system. The effects of mechanical stretch, as would occur during the development of ileus, were subsequently examined. The effects of CXCL1, an inflammatory mediator shown to increase in both animal models of ileus and in trauma patients with ileus, were also examined.

Results

HCN inhibition with ZD7288 suppressed agonist‐induced intestinal contractile activity in a dose‐dependent, tetrodotoxin‐resistant manner. Inhibition with an alternative HCN inhibitor, Zatebradine, inhibited agonist‐induced contractile activity in a similar manner. HCN inhibition suppressed calcium sensitivity in the small intestinal. Interestingly, HCN inhibition did not inhibit spontaneous contractile activity. Subjecting intestinal tissue to increased stretch or pretreatment with CXCL1 reduced the effects of ZD7288 on intestinal contractile activity, suggesting that both mechanical stretch and inflammation reduce HCN activity. Preliminary data show that increased intestinal wall stretch decreased HCN protein levels.

Conclusions

The effects of HCN inhibition are tetrodotoxin‐resistant; thus, the effects of HCN inhibition are mediated in the smooth muscle, not the enteric nervous system. Both mechanical stretch and inflammation, which occur during the development of ileus, can inhibit HCN activity. Taken together, these data show that HCN channels are important in agonist‐induced intestinal contractile activity. HCN channels are a novel potential target in the treatment of ileus in post‐operative or critically ill patients.

Support or Funding Information

Hungarian National Research, Development, and Innovation Office (NKFI 120669) and Institutional Developments for Enhancing Intelligent Specialization Grant (EFOP‐3.6.2‐16‐2017‐0006)
Hypergravity Reduces Astrocyte Migration by Altering Cytoskeletal Dynamics
Authors: Liemersdorf Christian , Lichterfeld Yannick , Kalinski Laura , Frett Timo , Jordan Jens , Hemmersbach Ruth
Published: April 2020

Glial scar formation through astrocytes impairs neural regeneration following spinal cord injury, head trauma, or stroke. Astrocyte migration towards the lesion and induction of a reactive astroglial phenotype require dynamic cytoskeletal protein rearrangements. In this glial cell model, hypergravity was used to alter cytoskeletal dynamics, i.e. stabilizing microtubules while destabilizing actin filaments. We hypothesize that increased gravitational (mechanical) loading by means of centrifugation (hypergravity) modulates in vitro astrocyte function in a way that could reduce their potential for scar formation.

We exposed primary murine cortical astrocytes to 2g using two types of custom‐designed hypergravity platforms at DLR (Cologne, Germany) and assessed a variety of parameters important for glial scarring in vitro. The platforms unlike commercial laboratory centrifuges model physiological hypergravity and allow for cell cultivation and live‐cell imaging. Primary astrocytes were isolated from wildtype (C57BL/6J) as well as transgenic LifeAct‐GFP mice and subjected to increased gravitational load. We measured cell proliferation and survival, after 7 days of exposure to 2g, as well as spreading and migration rate online for 24h. We visualized morphological features, cytoskeletal actin filament dynamics, reactivity markers and investigated expression levels of focal adhesion‐related proteins.

The exposure to 2g hypergravity induced a decrease in cell spreading (20%) coincidental with an inhibited migratory behavior (40%) and altered cytoskeletal dynamics. Astrocytic proliferation and survival were not affected. The expression of the focal adhesion marker vinculin was increased by 70–80%.

We conclude that hypergravity attenuates astrocyte spreading and migration. These parameters are crucial for glial scar formation, while basic cellular processes, such as proliferation and apoptosis were unchanged. The response appears to be mediated through altered cytoskeletal dynamics and may provide targets for therapies promoting neuronal regeneration.
Unraveling Intron Retention: Differential Expression Pattern in Brain and Kidney Cells After Hypoxia or Ischemia
Authors: Northrup Victoria , Perez Lester J. , Edgett Brittany A. , Simpson Jeremy A. , Brunt Keith R.
Published: April 2020

Introduction

The inclusion of introns in the mature mRNA (intron retention), has been recently uncovered as a mechanism for the regulation of genetic expression in vertebrates. Intron retention is implicated in tissue specific protein diversity, splicing regulation and control of gene expression. We have recently identified intron retention as a mechanism involved in the regulation of erythropoietin (EPO). EPO is the key regulator of erythropoiesis and is transcriptionally regulated by hypoxia inducible factor 2 (HIF2). The ability to easily detect intron retention could add to our ability to better understand genetic regulation of our genomes in stress conditions, such as ischemic diseases (ex/ myocardial infarction (MI) or stroke).

Objective

To evaluate the role of intron retention mechanism in EPO expression level to gain a better understanding of its regulation in response to hypoxia and ischemia, conditions which result in EPO expression.

Methods

HTB16 (neuroblastoma), CHP212 (glioblastoma), hCMEC/D3 (blood brain barrier) and HEK293 (kidney) were placed in hypoxic (1% oxygen), ischemic (1% oxygen, no serum or glucose) or normoxic conditions (20% oxygen) for 24 hours prior to RNA extraction. Quantitative polymerase chain reaction (qPCR) was performed with two sets of primers for EPO, the first set was targeting intron inclusion with each primer in a separate exon (intron primers) and the second set was targeting a region that spans two exons, which would not amplify if introns were present (non‐introns). Relative quantification for the level of expression was determined by qPCR using β‐actin and YWHAZ as reference genes for normalization purposes. Statistical analysis of transcript expression and the ratio between intron and non‐intron detecting primer sets were determined using analysis of variance (ANOVA).

Results

EPO mRNA level was increased in HTB16, CHP212 and HEK293, but not in hCMEC/D3 cell lines in response to ischemia, but not hypoxia. However, when the ration of intron vs. non‐intron expression level was determined a trend towards more intron retaining transcripts in HTB16, CHP212 and hCMEC/D3 but not in HEK293 was observed, suggesting an induction for the expression of this gene under this ischemic condition.

Conclusions

Intron retention mechanism in EPO is taking place in brain‐derived cell lines, but not in kidney‐derived cells. These results indicate a tissue specificity of intron retention mechanism that could regulate differential expression level of EPO under different physiological and stressful conditions. These findings highlight the complexity of EPO regulation. Hence, intron retention must be considered when examining the expression of a gene to better understand expression in ischemic disease models, such as MI or stroke.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC)
Effect of Natural Menopause and Hormone Therapy on Markers of Cellular Senescence
Authors: Faubion Laura , White Thomas A. , Peterson Brett J. , Singh Atul K. , Mielke Michelle M. , Miller Virginia M.
Published: April 2020

Background

Accumulation of senescent cells and the release of proteins from those cells contribute to aging‐related disorders. Estrogen may reduce physiological changes associated with aging through inhibition of cell senescence. This study aimed to determine the effects of menopausal hormone treatments on circulating levels of cell senescence markers.

Methods

GDF 15, TNF R1, FAS, and MIP1α were measured in serum using bead‐based assays and compared among women who were within 3 years of natural menopause (mean age 52 years) participating in the Kronos Early Estrogen Prevention Study (KEEPS). Women were randomized to either placebo (n=38), oral conjugated equine estrogen (oCEE, n=37), or transdermal 17β‐estradiol (tE2, n=34). Serum levels of the senescent markers for each treatment group were compared to placebo 36 months after randomization using the Wilcoxon rank‐sum test.

Results

Serum levels of GDF 15, TNF R1, and FAS but not MIP1α were lower in both the oCEE and tE2 groups compared to placebo. However, the difference in levels between treatment and placebo were greater for oCEE [−108 pg/mL (p=0.008), −234 pg/mL (p=0.0006), and −1374 pg/mL (p<.0001), respectively] than for tE2 [−76 pg/mL (p=0.072), −105 pg/mL (p=0.076), and −695 pg/mL (p=0.036), respectively].

Conclusions

In recently menopausal women, oCEE and tE2 reduced circulating levels of some markers of cell senescence. Differences in the magnitude of effect may reflect the differences in circulating levels of 17β estradiol, estrone, and sulfonated estrogens between the two active treatments.

Support or Funding Information

The research is supported by the Kronos Early Estrogen Prevention Study, NIA U54‐AG44170, and the Mayo Fnd.
The Influence of Healthy Aging on K+ Channel‐Dependent Microvascular Dilatory Function
Authors: Serviente Corinna , Berry Craig , Kenney W. Larry , Alexander Lacy M.
Published: April 2020

Aging is associated with reduced microvascular endothelial function, contributing to increased cardiovascular disease risk. Microvascular dysfunction is usually measured by evaluating endothelial nitric oxide‐dependent vasodilation. However, other vascular signaling pathways, including endothelial derived hyperpolarizing factors (EDHFs), acting through K+ channels, are also important for maintenance of vascular health and may be influenced by age and disease. It remains unclear how microvascular EDHFs are affected by healthy aging.

PURPOSE

To evaluate the microvascular endothelial reliance on K+ channel‐mediated dilation with healthy aging.

METHODS

Healthy younger (n=6, 27±2 yrs, 23.4±0.5 kg/m2, 40.9±3.9 ml/kg/min) and older (n=9, 64±2 yrs, 23.7±1.1 kg/m2, 37.5±4.0 ml/kg/min) men and women (younger: 3M/3W, older: 3M/6W) underwent graded infusions of the endothelial dependent agonist acetylcholine (ACh, 10−6 to 10−2 M) alone and in combination with the K+ channel blocker tetraethylammonium (TEA, 25 mM) via cutaneous intradermal microdialysis. Additionally, responses to graded infusions (20mM to 100mM) of the K+ channel activator Na2S were evaluated (older, n=10). Red blood cell flux was measured at each site using laser Doppler flowmetry. Cutaneous vascular conductance (CVC: flux/mean arterial pressure) was calculated as a percentage of site specific maximum (43°C + 28mM sodium nitroprusside). Data are presented as mean ± SEM.

RESULTS

There was a main effect of age (p<0.001), such that older adults had reduced overall %CVCmax compared to younger adults. There was an interaction between site treatment and age (p<0.001). Within older adults, blood flow was lower in the TEA vs. the ACh site starting at 10−5 M (ACh: 31.8±4.1%CVCmax vs. TEA: 13.2±4.1%CVCmax, p=0.0031), while differences were not observed in younger adults until 10−4 M (ACh: 51.5±5.0%CVCmax vs. TEA: 35.5±5.0%CVCmax, p=0.028). In response to Na2S, there was a main effect of age (p=0.032), with older adults showing overall higher %CVCmax compared to younger adults (older: 56.1±4.4%CVCmax vs. younger 38.8±5.7%CVCmax).

CONCLUSIONS

These data suggest that heathy older adults have increased reliance on K+ channel‐dependent endothelial dilatory mechanisms compared to younger adults, suggesting increased reliance on EDHFs with healthy age.

Support or Funding Information

NIA T32‐AG049676
Insights into SARM1’s Enzymatic Mechanisms for Executing Pathological Neuronal Death
Authors: Loring Heather , Thompson Paul
Published: April 2020

The novel NAD+ glycohydrolase, sterile alpha and toll/interleukin receptor (TIR) motif‐containing protein 1 (SARM1), is an active executioner in progressive axonal and neuronal degeneration. This type of degeneration, termed Wallerian degeneration, defines a number of diseases, including neuropathies, traumatic brain injury and neurodegenerative diseases. However, no therapies exist to mitigate these diseases. In fact, prior to the recent discovery of SARM1’s role in triggering Wallerian degeneration, the process was believed to occur passively. Stimulation of SARM1 activates its intrinsic NAD+ hydrolase activity, resulting in NAD+ cleavage and neuronal death. By contrast, knock down or knock out of SARM1 delays degeneration and has shown therapeutic efficacy for various Wallerian‐type diseases, including peripheral neuropathy and traumatic brain injury. NAD+ supplementation alone is also sufficient to delay degeneration, indicating that inhibiting SARM1‐mediated NAD+ hydrolysis would be efficacious. Thus, SARM1’s pivotal role in Wallerian degeneration makes it an attractive therapeutic target that could prevent disease progression. However, the design of therapeutics targeting SARM1 is limited by the dearth of knowledge surrounding its inherent NAD+ hydrolase activity. Illuminating this black box has also been hindered by technical difficulties in obtaining pure active protein. Recently, we were able to purify active bacterially expressed protein, yielding insights into SARM1 morphology and cellular mechanism. Furthermore, we have performed detailed kinetic, catalytic and inhibitory studies, to determine SARM1’s kinetic and catalytic mechanisms and identify potential inhibitors to block SARM1‐mediated cell death. The results of these analyses will be discussed.

Support or Funding Information

NIH NINDS F31‐NS‐108610‐01
Sex Differences and Inactivity In Rats Determine the Progression of Increased Sympathetic Activity Over Time.
Authors: Morrison Paul , Tan Can , Draghici Adina , Mueller Patrick
Published: April 2020

Cardiovascular disease (CVD) is the leading cause of death, with males having a higher risk than females; that is, until menopause, when the risks reverse. Risk factors for CVD include a sedentary lifestyle as well as elevated sympathetic nerve activity (SNA). In women, resting SNA is influenced by the menstrual cycle; however, the interaction between sedentary conditions and the presence or absence of the female reproductive cycle on baseline sympathetic nerve activity is unknown. Therefore, the purpose of this study was to determine the effects of sedentary versus active conditions on resting SNA in male and female rats over time. In particular, we studied rats at four and eight weeks of age (i.e. before and after the onset of the estrous cycle, respectively in female rats). We hypothesized that time‐dependent increases in SNA in sedentary male rats would be offset by four weeks of voluntary exercise (running wheels) and attenuated in sedentary female rats due to the onset of the estrous cycle. We recorded splanchnic SNA from 32 rats under Inactin anesthesia and determined SNA action potential frequency according to Tan et al., (2009). Using a two‐way analysis of variance (ANOVA), with sex and condition as main effects, we found that four week‐old male rats exhibited lower SNA compared to eight week‐old sedentary males (42.0 ±6.3 Hz vs. 62.4 ±5.8 Hz; p=0.015; n=6 each), while exhibiting no significant difference from eight week‐old active males (37.4 ±6.1 Hz; p=0.003; n=7). In contrast, SNA was not significantly different between four week‐old females (54.6 ±4.5 Hz; n=4) and eight week‐old sedentary females (46.0 ±4.3 Hz; p=0.348; n=5), suggesting a lack of inactivity‐related increases in SNA as observed in males. SNA in four week‐old females was also not significantly different than eight week‐old active females (51.0 ±5.8 Hz; p=0.708; n=4). Our results suggest a sedentary lifestyle promotes increases in SNA over time in males but not females, due to physical inactivity in males and a protective effect of ovarian hormones in females. Male sex‐ and inactivity‐dependent increases in SNA likely play an important role in the higher risks of CVD observed in males compared to females.

Support or Funding Information

(NIH HL096787‐08; AHA25810010)
GPCR Kinase 2 (GRK2) Phosphosphorylation of the Alpha 2A Adrenergic Receptor in Intact Cells
Authors: Sterne-Marr Rachel , Barnett Jessica , Hebert Aaron , Weldin Erica , Farina Zachary , Guirguis Jeska , Varney Matthew
Published: April 2020

The α2A‐adrenergic receptor (α2A‐AR) is activated by the catecholamine norepinephrine and plays a variety of roles including neuromodulation, vasoconstriction in the cardiovascular system, regulation of insulin secretion and platelet aggregation. Platelet α2A‐AR serves as an excellent substrate for agonist‐dependent phosphorylation by G protein‐coupled receptor (GPCR) kinase 2 (GRK2) in vitro. α2A‐AR is structurally distinct from the well‐characterized β2‐adrenergic receptor (β2AR) in possessing a large (~100 amino acid) third intracellular loop containing four successive serine residues that serve as phospho‐acceptors. When overexpressed with GRK2, the receptor is phosphorylated at all 4 phospho‐sites. Our long‐term goal is to understand how GRK2 interacts with and is activated by GPCRs. We have mapped residues on GRK2 that are required for phosphorylation of β2AR and recruitment to α2A‐AR in intact cells. We wish to assay phosphorylation of α2A‐AR in intact cells and because no commercially available α2A‐AR phosphorylation site antibodies currently exist, we set out to develop such a reagent. An antibody that recognizes α2A‐AR phosphorylation at serines 296–299 was generated and characterized. This antibody detects an epinephrine‐stimulated signal that is increased two‐fold by transfection of GRK2 in COS‐7 cells. We also found that the α2A‐AR was phosphorylated relatively well in the absence of epinephrine and transfected GRK2 suggesting that α2A‐AR is a good substrate for an endogenous COS‐7 cell kinase. Mutagenesis of the receptor has allowed us to measure agonist‐ and GRK‐dependent phosphorylation of α2A‐AR. We are now in a position to characterize GRK2 phosphorylation of this receptor.

Support or Funding Information

This work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).