bluuebunny/crossref_metadata_2025 huggingface dataset:
Coronary Smooth Muscle Cytodifferentiation is Associated with an Increase in Pro‐Inflammatory, Pro‐Calcifying Gene Expression in an Organ Culture Model of Coronary Artery Disease
Authors: Badin Jill , Sturek Michael
Published: June 2021

Introduction

During coronary artery disease (CAD) progression, coronary smooth muscle (CSM) cells dedifferentiate into synthetic or osteogenic phenotypes that induce CSM proliferation and vascular calcification, respectively. This phenotypic switching is in parallel to the upregulation of intracellular [Ca2+]i handling observed in early CAD and downregulation observed in late CAD. These [Ca2+]i handling alterations may play a role in promoting coronary artery calcification (CAC), which is associated with increased risk of cardiovascular health complications and mortality. Therefore, the aims of this study are two‐fold: 1) utilize an in vitro organ culture model of CAD to elucidate the extent of CSM phenotypic switching and 2) identify what genes are upregulated in each major CSM phenotype. Collectively, the data show possible signaling mechanisms and cellular processes that contribute to CAD pathophysiology, specifically CAC.

Methods

Coronary rings from lean Ossabaw miniature swine were either used fresh or cultured in osteogenic media containing 10% FBS, 180 mg/dL glucose, 1% penicillin/streptomycin, 3.8 mM inorganic phosphate, and 7.5 U/mL alkaline phosphatase in DMEM for 3 days. To identify cells, cell population heterogeneity, and gene expression, CSM were enzymatically dispersed and used for single‐cell RNA‐sequencing.

Results

The presence of differentiated, contractile CSM phenotypic markers including actin (ACTA2), myosin light chain (MYL9), myosin heavy chain (MYH11), and calponin (CNN1) were drastically reduced in the transcriptome of cells in cultured arterial rings. CSM from cultured rings also exhibited higher levels of transforming growth factor beta 1 (TGFB1) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin (SMARCA4), both markers of the synthetic, proliferative phenotype. Furthermore, genes related to inflammation, chemotaxis, oxidative stress, extracellular matrix degradation, and vascular calcification were upregulated in CSM from cultured arterial rings when compared to CSM from fresh, noncultured rings.

Conclusion

After 3 days cultured in osteogenic media, CSM in whole arterial rings undergo dedifferentiation, as evidenced by decreased contractile markers and increased proliferative markers. This is associated with the upregulation of several genes associated with CAD pathophysiology, including genes involved in inflammation, calcification, and oxidative stress. This study will further help to parse out the complex cellular mechanisms involved in the progression of CAD and CAC.

Support or Funding Information

This work was supported by the CorVus Biomedical Research Fund.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CK2 Phosphorylation of ENaC Directs Ankyrin‐3 Activity
Authors: Berman Jonathan Max , Mironova Elena , Stockand James , Archer Crystal
Published: June 2021

The Epithelial Na+ Channel, ENaC, is the final arbiter of sodium excretion in the kidneys. As such, discretionary control of ENaC by hormones is critical to the fine‐tuning of electrolyte and water excretion and consequently, blood pressure. We recently showed that phosphorylation of ENaC by CK2 in a putative ANK3 binding domain regulates ENaC activity. Therefore we tested the necessity and sufficiency of ANK3 for normal ENaC function. We produced tissue specific ANK3 knockout mice in the aldosterone sensitive distal nephron. Knockout was verified by immunohistochemistry. Split open tubule recordings from these mice showed significantly decreased baseline ENaC activity. The mice displayed a insensitivity to the specific CK2 inhibitor TBB, a casein‐kinase II inhibitor compared to control. Together these results suggest that ANK3 modulates ENaC activity by interacting with CK2 phosphorylation sites in Beta and Gamma ENaC. In future work we will examine the mechanism by which this interaction modifies ENaC activity.

Support or Funding Information

This research was supported by American Heart Association Grants 15GRNT‐22930030 and 17GR‐NT‐3292002 (to J. D. Stockand) and 17POST33660468 (to J. M. Berman) and by National Institutes of Health Grants T32‐HL‐00744536A1 (to J. D. Stockand and J. M. Berman), K12GM111726 (to J. M. Berman), and F32‐DK‐104572 (to E. Mironova).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA Seq Analysis Reveals Metabolic and Natriuretic Pathways Regulated by Renal T Cell Infiltration
Authors: Dayton Alex , Yang Chun , Alsheikh Ammar , Abais‐Battad Justine , Bukowy John , Pan Xiaoqing , Mattson David , Cowley Allen , Evans Louise
Published: June 2021

Previously, we presented work using a servo‐control system to maintain pressure to the left kidney at normotensive levels during high salt feeding. Using this approach we have demonstrated that T‐cell infiltration into the kidney during salt‐induced hypertension is a direct consequence of pressure. Further, we used RNA sequence data to detect transcriptional pathways that differ between hypertensive and normotensive kidneys, including several pathways related to immune cell migration and activation.

In order to further study the interactions between T‐cells and salt‐sensitive hypertension in the Dahl S rat, we use a CD247−/− strain, which lack functional T‐cells. These rats demonstrate reduced pressures relative to wild‐type (WT) rats when fed high salt. Here, we present a study in which we adapted the servo‐control method so that the renal perfusion pressure to the left kidney of WT rats was matched on a daily basis to that of the left kidney of CD247−/− rats. This results in exposure of the left kidneys to an equivalent hypertensive response to salt. Analysis of these “pressure‐matched” kidneys therefore controls for the potentially confounding effects of pressure, and allows for a more direct comparison of tissues to reveal the effects of renal T‐cell infiltration in hypertension.

Both CD247−/− and WT rats (N=4) were instrumented for servo‐control, as described previously. These rats were then fed a 4% NaCl diet to induce hypertension. Using the servo‐control system, the left kidney of the WT rats was maintained on a daily basis to match the blood pressures exhibited by the CD247−/− rats. At the conclusion of 12 days, the rat's kidneys were flushed with saline, followed by isolation of the renal medulla for RNA‐seq analysis.

RNA was reverse transcribed and then sequenced on an Illumina Hi‐Seq 2500 sequencer. Data was then aligned to the RN6 genome and quantified. 2865 transcripts were found to be significantly different between the CD247−/− and WT medullas (p < 0.05). Overrepresentation analysis using DAVID found significant differences in transcripts belonging to oxidative phosphorylation (p < 8.1 * 10−22), TCA cycle (p < 5.0 * 10 −9), collecting duct acid secretion (p < 5.7 * 10 −4) and aldosterone‐regulated sodium reabsorption (p < 0.013) pathways, among others. This analysis suggests that these pathways are regulated by T‐cell infiltration and activation within the renal medulla. These pathways are viable targets for the further study of the effect of immune cells on blood pressure.

Support or Funding Information

NIH F30HL127979 AHA 17SDG33660574

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single Cell Ablation in the Zebrafish Kidney using SPIM and a Bessel Beam
Authors: Brown Cara , Buckley Charlotte , Hoffmann Scott , Rider Sebastien , Girkin John , Mullins John
Published: June 2021

Selective plane illumination microscopy (SPIM) uses low‐powered laser light to rapidly and precisely capture high‐resolution images of cells within live organisms, revealing fine details of the cell structure. By combining a bespoke SPIM/Bessel beam in combination with appropriate fluorescent cell‐specific reporters, individual cells can be located and targeted for ablation via apoptosis.

Incorporation of fluorescent reporters such as LifeAct‐red fluorescent protein (RFP) or the photosensitizing protein, KillerRed (KR), into the genome of the optically clear zebrafish larvae, allows specifically labelled cells to be visualized and/or optically ablated deep within developing organs and in real‐time. We have used this technology to visualize and target a) cells of the renin lineage (CoRLs)1 and b) podocytes, within the live Zebrafish kidney. Transgenic fish expressing KR under the control of the renin and podocin promoters were established and used independently for imaging/ablation experiments. Using a Bessel beam in combination with SPIM to precisely activate KR allows relatively low laser power to be employed resulting in 100% viability of the fish following laser treatment. Low energy laser ablation allows the cell injury/ablation to be imaged and assessed over an extensive time period (hours).

Support or Funding Information

BHF Center of Research Excellence Award; Kidney Research UK award; EPSRC/MRC DT (Optima) Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Ketogenic Diet on p70S6k Phosphorylation in Young Adult and Old Soleus Muscle
Authors: Bennett Shantelle L.H. , Smith Andrew H. , Ramirez J. Lucas , Thomson David M.
Published: June 2021

A ketogenic diet (KD) has been proposed as a treatment for many conditions including obesity, diabetes, epilepsy, and Parkinson's Disease. Various studies have shown its effects on health and lifespan in model organisms suggesting that it is beneficial for many individuals. Some concern exists that KD might negatively impact anabolic signaling through the mTOR pathway, but the molecular signaling effects of the diet remain largely unexplored, particularly in skeletal muscle. The purpose of this study, therefore, was to determine how KD affects downstream mTOR signaling in skeletal muscle from young and old rats. Young adult (YA; 5 mo.) and old (O; 28 mo.) male Fisher 344 rats were isocalorically fed either a ketogenic diet (KD; Envigo Teklad Custom Diet, TD.10911; 22.4% protein, 77.1% fat, 0.5% carbohydrates) or standard diet (STD; Envigo Teklad Rodent Diet, 8604; 32% protein, 14% fat, 54% carbohydrate) for four weeks. Soleus muscles were harvested and assessed for muscle size and effects on p70 ribosomal protein S6 kinase (S6k) phosphorylation. Soleus muscles from YA KD rats were ~11% smaller than for corresponding YA STD rats, and O KD soleus muscles were ~6% larger vs. O STD muscles, though neither of these differences were significant. Phosphorylation of S6k was significantly (p < 0.05) decreased 75% in YA KD vs. YA STD. S6k phosphorylation was reduced 88% in O muscles, but, in contrast to the YA muscles, was increased by ~400% by KD vs. STD in O muscles. Based on this evidence, we conclude that in slow‐twitch soleus muscles, administration of KD can decrease skeletal muscle mTOR signaling in YA muscles and increase its signaling in O muscles.

Support or Funding Information

This work was supported by a Brigham Young University Gerontology Program award.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence of endothelial‐to‐mesenchymal transition in an animal model of pulmonary arterial hypertension
Authors: Morrow K. Adam , Brown Jonathan R. , Morrow Rebekah L. , Snyder Emily A. , Vasauskas Audrey A
Published: June 2021

Pulmonary arterial hypertension (PAH), a disease of the small pulmonary arteries, is characterized by pulmonary vasoconstriction, vascular cell proliferation, and vascular remodeling leading to right ventricular hypertrophy and failure. The remodeled vessels exhibit signs of endothelial layer damage and smooth muscle cell proliferation. It is also well‐established that mutations in the bone morphogenetic protein receptor 2 (BMPR2) predispose individuals to development of PAH. Recent studies in PAH mouse models and in cultured human pulmonary artery endothelial cells (where BMPR2 has been experimentally knocked down) suggest that endothelial cells involved in the vascular remodeling of PAH undergo transdifferentiation. Here, we show that pulmonary artery endothelial cells (PAECs) isolated from the rat Sugen (SU516)/hypoxia model of pulmonary arterial hypertension exhibit phenotypic changes characteristic of endothelial‐to‐mesenchymal transition (EndMT). Preliminary data also suggest decreased BMPR2 expression in the PAH model PAECs. Cells were obtained from the University of South Alabama Center for Lung Biology (CLB). Briefly, Fisher 344 rats were injected with SU5416 (20mg/kg) or vehicle control and exposed to hypoxia (10% O2 or normoxia for control animals) for three weeks and returned to normoxia for at least two weeks. PAECs were harvested using established CLB protocol. PAECs from the PAH animals (SURAT) exhibited a significant increase in alpha smooth muscle actin (αSMA) expression as compared to the control PAECs, as evidenced by immunocytochemistry and real time PCR. These cells retained expression of endothelial cell markers von Willebrand Factor (vWF) and platelet endothelial cell adhesion molecule (PECAM‐1). However, expression of both PECAM‐1 and vWF was decreased in the SURAT cells compared to control PAECs. Expression of tight junctional protein‐1, vascular adhesion molecule‐1 (VCAM‐1), intracellular adhesion molecule‐1 (ICAM‐1), claudin‐1, and selectin was also decreased in the SURAT PAECs as compared to control cells. These data suggest that PAECS exhibit signs of cellular reprogramming in Sugen/hypoxia‐treated animals, and also provide insight into a cellular model that directly correlates with a well‐established animal model of PAH.

Support or Funding Information

Supported by NIH1R15HL137135‐01A1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LDLR‐Related Protein 1 Increases Cytokine Sensitivity – Implications for Recovery after Brain Damage
Authors: Ahmad Sadiya Noor , Reed Pam , Sprague Shane , Sayre Naomi
Published: June 2021

The limited treatment options for ischemic stroke patients have resulted in stroke being a leading cause of death and the primary cause of long‐term disability in the U.S. Finding effective treatment options requires a better fundamental understanding of the ongoing processes that contribute to poor long‐term outcome.

Expression of Apolipoprotein E4 predisposes stroke patients to poor long‐term outcome. This study aims to test one possible mechanism by which ApoE4 contributes to cognitive decline after stroke. Here, we examine the effect of a major ApoE4 receptor, low‐density lipoprotein receptor related protein 1 (LRP1) on sensitivity to stress in astrocytes.

LRP1 binds and moves extracellular ligands and plasma membrane proteins into the endocytic system. Others have shown that LRP1 regulates cell‐surface TNF receptor (TNFR1) in non‐astrocytic cells. We propose That LRP1 similarly regulates TNFR1 in the central nervous system to attenuate inflammatory response after stroke. Studies have shown that ApoE4 slows the recycling of endocytic LDL receptors. We hypothesize that ApoE4 inhibits the ability of LRP1 to remove TNFR1 from the plasma membrane. This is expected to increase cytokine sensitivity, resulting in worse outcome after stroke.

We investigated the effect of LRP1 on astrocyte TNFα signaling and response in immortalized ApoE null mouse astrocytes subjected to lentiviral‐mediated knockdown of LRP1. The astrocyte response to TNFα stimulation was tested in a time dependent manner using Western blotting of NFkB pathway components, which are the downstream mediators of TNFα signaling. We also tested astrocyte viability after prolonged TNFα stimulation using Alamar Blue reagent. We found that LRP1 deficient cells have increased phosphorylation of NFkB upon TNFα stimulation, and that loss of LRP1 resulted in significant loss of astrocyte viability after prolonged stimulation. Altogether, our results indicate that loss of LRP1 renders astrocytes more sensitive to TNFα. Future experiments will focus on testing the influence of LRP1 on recovery after middle cerebral artery occlusion in mice.

These studies will elucidate how astrocyte‐LRP1 contributes to outcome after stroke, and helps us to understand one potential way that ApoE4 exerts pathological effects. A better understanding of the long‐term processes after stroke will allow identification of therapies which improve the morbidity and mortality associated with stroke.

Support or Funding Information

Institutional National Research Service Award, T32 NIH/NCATS, TL1TR002647, funded by National Institutes of Health/National Center for Advancing Translational Sciences

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tunicamycin‐induced ER Stress Effect on Cardiac Contractility
Authors: Alabdaljabar Mohamad Saleh , Valencia Sara Osorio , Han Young Soo , Sieck Gary C.
Published: June 2021

In cardiomyocytes, the endoplasmic reticulum (ER) plays a dynamic role in excitation‐contraction coupling by regulating cytosolic Ca2+ concentration ([Ca2+]cyt). Under pathological conditions the ER accumulates unfolded proteins triggering a homeostatic ER stress response to minimize and limit protein unfolding (via chaperone proteins) and accumulation (by decreasing synthesis and increasing degradation). The interactions between ER stress, [Ca2+]cyt regulation and downstream contractile responses remain unknown, although such interactions may underlie the etiology of various cardiovascular diseases. We hypothesized that in cardiomyocytes ER stress alters the regulation of [Ca2+]cyt and reduces cardiac contractility by decreasing myofilament Ca2+ sensitivity. In isolated rat cardiomyocytes, ER stress was induced by tunicamycin, a naturally occurring antibiotic that induces ER stress by inhibiting N‐linked glycosylation. In one set of experiments, we used Western blotting techniques to evaluate the effects of ER stress on phosphorylation of cTnI at Ser23/24. In a second set of experiments, we evaluated the impact of tunicamycin on excitation‐contraction coupling by simultaneously measuring [Ca2+]cyt (using Fura‐2 AM ratiometric imaging) and contractile (sarcomere length (SL) shortening) responses to electrical field stimulation using an IonOptix system. Tunicamycin induced an increase in phosphorylation of cTnI by 3–6 h. The evoked [Ca2+]cyt transient was relatively unaffected by tunicamycin while the contractile response was markedly reduced. This resulted in a rightward shift in the phase loop relationship between [Ca2+]cyt and SL shortening indicating reduced myofilament Ca2+ sensitivity. We conclude that ER stress may underlie contractile dysfunction in a variety of cardiac diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of IL‐10 in Bone‐Marrow Derived Macrophages by Helicobacter Hepaticus Supernatant is Mediated by Gpr109a
Authors: Al‐odaini Asma , Singh Nagendra
Published: June 2021

Interleukin 10 (IL‐10) is an anti‐inflammatory cytokine, and its polymorphism is linked to inflammatory bowel diseases (IBDs) in humans. Interactions between gut microbiota and the host's the immune cells play a key role in shaping immune responses and intestinal health. Helicobacter hepaticus is a member of mouse gut microbiota that is tolerated by the host but can induce colonic inflammation in mice in absence of an intact IL‐10 signaling. H. hepaticus secretes a polysaccharide that signals through Toll‐like receptor 2 (TLR2) to induce IL‐10 production. Gpr109a is a G‐protein coupled receptor that is expressed at a very high level on innate immune cells, and it is thought to interact with gut microbe‐derived molecules to induce production of anti‐inflammatory cytokines and suppress colonic inflammation. It is hypothesized that H. hepaticus induction of IL‐10 through TLR2 is mediated by Gpr109a. To test whether Gpr109a is involved in IL‐10 induction by H. hepaticus secretions, bone‐marrow derived macrophages (BMDMs) of WT and Gpr109a KO mice were stimulated by H. hepaticus supernatant then IL‐10 mRNA and protein levels were determined. WT BMDMs were found to produce significantly more IL‐10 mRNA and protein compared to Gpr109a KO BMDMs. In addition, ERK1/2 is a protein complex that gets activated when the TLR2 is stimulated by H. hepaticus to induce IL‐10. We tested whether ERK1/2 activation by H. hepaticus supernatant is dependent on Gpr109a by using an immunoprecipitation assay for total and pERK1/2 upon stimulation with H. hepaticus supernatant for WT and Gpr109a KO BMDMs. Our data suggests that Gpr109a plays a critical role in activation of ERK1/2 downstream of TLR2 signaling leading to induction of IL‐10. These findings will be of a value in delineating the molecular mechanism of Gpr109a signaling and investigating Gpr109a role in preventing H. hepaticus induced colitis and intestinal cancers.

Support or Funding Information

This projected was funded by Augusta University (The STAR Program) and performed in Dr. Nagendra Singh's laboratory and under his supervision with technical training and support by Brinda Bhatt and Dr. Huabin Zhu.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aerobic Training Reduces Immune Cell Recruitment and Cytokine Levels in Adipose Tissue in Obese Mice
Authors: Silva Albená Nunes , Lacerda Debora Romualdo , Moraes Michele Macedo , Costa Kátia Anunciação , Rodrigues Débora Fernandes , Sabino Josiana Lopes , de Souza Cordeiro Letícia Maria , Pinho Vanessa , Teixeira Mauro Martins , Wanner Samuel Penna , Soares Danusa Dias , Ferreira Adaliene Versiani Matos
Published: June 2021

Obesity is associated with an energy imbalance that results from excessive energy intake, low diet quality and a sedentary lifestyle. In this regard, the increased consumption of a high‐refined carbohydrate diet (HC) is strongly related to higher adiposity and low‐grade inflammation. Aerobic training is a well‐known non‐pharmacological intervention to treat obesity and metabolic disturbances. However, the mechanisms through which aerobic training ameliorates the low‐grade inflammation induced by the HC diet need to be further investigated. Herein, our hypothesis was that aerobic training would decrease the recruitment of leukocytes in the adipose tissue thereby reducing the levels of cytokines and improving metabolism in mice fed the HC diet. Male Balb/c were assigned to the following groups: control non‐trained (C‐NT), control trained (C‐T), HC‐NT and HC‐T. Mice were submitted to moderate‐intensity training sessions that consisted of running 60 min/day for 8 weeks. The intravital microscopy technique was performed in vivo in anesthetized mice to visualize the microvasculature of the adipose tissue. The HC diet induced obesity and increased the influx of immune cells into the adipose tissue. In contrast, HC‐T mice presented a lower adiposity and adipocyte area. Furthermore, HC‐T mice showed an increased resting energy expenditure, a decreased recruitment of immune cells in the adipose tissue, reduced cytokine levels, and ameliorated hyperglycemia and fatty liver deposition relative to HC‐NT mice. Collectively, our data enhance the understanding about the anti‐inflammatory effect of aerobic training and shed light on the adipose tissue‐mediated mechanisms by which training promotes a healthier metabolic profile.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resveratrol protects UVB‐irradiated keratinocytes by upregulating HO‐1 expression and improving mitochondrial function via endogenous CO signaling
Authors: Averilla Janice Nullan , Oh Jisun , Kim Jong‐Sang
Published: June 2021

Excessive exposure to ultraviolet (UV) radiation has been reported to promote uncontrolled reactive oxygen species (ROS) production, particularly in the mitochondria, and thus implicated in the prevalence of skin injuries. Considering the antioxidative effect of resveratrol (RES) in reducing UV‐induced ROS production in human keratinocytes (HaCaT cells) and the role of carbon monoxide (CO) in regulating oxidative stress, we hypothesized that RES may promote cellular antioxidant response by activating heme oxygenase‐1 (HO‐1) which can generate its enzymatic product, CO, in UVB‐irradiated keratinocytes. To test our hypothesis, we treated HaCaT cells with CO‐releasing molecule 2 (CORM‐2) (source of CO) and RES either independently or in combination and irradiated the cells with UVB. Co‐treatment of CO with RES upregulated HO‐1 expression and subsequently reduced intracellular ROS in response to UV irradiation. Moreover, UVB treatment alone induced mitochondrial biogenesis, but treatment with CO and RES did not increase it further. With UVB treatment, RES showed enhanced mitochondrial respiration while CO did not instigate any effect; however, independent or co‐treatment mitigated mitochondrial ROS production. Taken together, our data support the impression that endogenous CO generated from RES‐mediated activation of HO‐1 may play a crucial role in cellular antioxidant response and mitochondrial function.

Support or Funding Information

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (IPET) through the High Value‐Added Food Technology Development Program, funded by the Ministry of Agriculture, Food, and Rural Affairs (Grant No. 116026‐03‐2‐HD020), Republic of Korea.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Effects of a Bout of Moderate‐intensity Physical Exercise on Adipose Tissue Inflammation in Lean and in Obese Mice
Authors: Silva Albená Nunes , Lacerda Debora Romualdo , Rodrigues Débora Fernandes , Silveira Ana Leticia Malheiros , Moraes Michele Macedo , Pinho Vanessa R , Teixeira Mauro Martins , Wanner Samuel Penna , Soares Danusa Dias , Ferreira Adaliene Versiani
Published: June 2021

Inflammation is an adaptive response that is triggered by the noxious stimuli in order to maintain the homeostasis. Previous studies have shown that higher lipolysis rate after fasting is associated with the influx of leukocytes and increased cytokine levels in the epididymal adipose tissue (EAT). Fat mobilization is also highly activated during and post‐exercise. In spite of substantial evidences having supported the link between inflammation and exercise, the association between EAT, cytokines and acute exercise is less explored. Herein, we characterized the EAT inflammation following exercise in lean and obese mice. We used in vivo confocal imaging from EAT vessel to display the higher recruitment of neutrophils 0 and 6 hours post‐exercise in lean mice. Additionally, we found that exercise enhanced the levels of IL‐6 and TNF‐alpha in the EAT which were correlated with increased serum free fatty acids in lean mice. Interestingly, obese mice subjected to exercise showed lower pro‐inflammatory level of cytokines in the EAT and lost the correlation between IL‐6, TNF‐alpha and free fatty acids. Furthermore, obese mice increased IL‐10 24h post‐exercise. Together, our data show that exercise induces inflammation in the EAT in lean mice. These responses may have a physiological purpose and be important to stimulate fat breakdown. Conversely, exercise in obese mice promoted a reduction in pro‐inflammatory mediators and elevated IL‐10. Despite of the low‐grade inflammation being associated with metabolic dysfunctions, at least in part, the inflammatory milieu in EAT may be also important to regulate fat remodeling induced by exercise.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IIntracellular BiP/GRP78 Mediates Endothelial Cell Permeability and Inflammation Associated with Acute Lung Injury
Authors: Leonard Antony , Su Pei‐Yi , Millar Michelle , Dubey Nikhil , Yule David , Rahman Arshad , Fazal Fabeha
Published: June 2021

The role of endoplasmic reticulum (ER) chaperone and signaling regulator BiP/GRP78 in acute inflammatory injury, particularly in the context of lung endothelium, is poorly defined. Recent studies have shown that BiP/GRP78 not only resides in the ER but is present in extra‐ER regions as well, and perform different functions. In this study, we monitored the effect of intracellular versus cell surface BiP/GRP78 in EC permeability and inflammation associated with acute lung injury (ALI). Studies were performed using SubAB, a holoenzyme that cleaves and specifically inactivates intracellular BiP/GRP78 versus SubA, which cleaves and inactivates only the cell surface BiP/GRP78. Stimulation of endothelial cell (EC) with proinflammatory mediator thrombin showed a characteristic time‐dependent change in TER (trans‐endothelial resistance), a real time measurement of EC permeability, which was protected in the presence of SubAB but not in the presence of SubA and its inactive mutant SubAA272B. In order to further dissect the mechanism of thrombin‐mediated EC permeability, we determined the role of BiP/GRP78 in mediating Ca2+ signaling, a critical determinant of EC permeability. Interestingly, SubAB significantly reduced thrombin‐induced Ca2+ release from the ER stores as well as Ca2+ entry from the extracellular medium; however SubA and SubAA272B failed to mediate the above response. Furthermore, mechanistic analysis revealed that thrombin‐induced activation of STIM‐1 (Stromal Interaction Molecule 1) via Y361 phosphorylation, which stimulates Ca2+ entry across the plasma membrane, was blocked in the presence of SubAB. In addition, thrombin‐induced phosphorylation of eNOS at serine 1177 as well as the association of phosphorylated eNOS with p190GAP, which is a major mechanism responsible for AJ disassembly resulting in increased EC permeability, was significantly blocked by SubAB. Interestingly, thrombin‐induced inflammatory responses were not affected in the presence of SubA as opposed to SubAB. Together, these data highlight a novel role of intracellular BiP/GRP78 in mediating EC barrier disruption and inflammation associated with ALI

Support or Funding Information

NIH (HL096907 and HL130870)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Co‐localization of Isolectin B4 (IB4), Ionized Calcium‐Binding Adapter Molecule 1 (Iba1), and Von Willebrand Factor (vWF) Immunostaining in the Mid‐Cervical Spinal Cord After Spinal Injury
Authors: Fusco Anna F , Sunshine Michael D , Streeter Kristi A , Wollman Lila B , Jorgensen Marda L , Fuller David D
Published: June 2021

IB4 immunostaining is often used to histologically identify thin fiber non‐peptidergic afferent neurons in the spinal cord. However, IB4 can also identify both endothelial cells and microglia. In studies of adult Sprague Dawley rats with chronic (12 weeks) cervical (C2) hemisection injury (Hx; N=10), we noted robust IB4 immunostaining in mid‐cervical dorsal root ganglia cell bodies as well as spinal cord lamina II, indicating labeling of thin fiber afferents. We also observed distinct clusters of IB4+ cells in the mid‐cervical spinal cord ipsilateral to Hx in the area of the corticospinal tract (N=6 of 10 rats), and in ventrolateral (N=9 of 10) and ventromedial white matter (N=9 of 10). Incubation of tissues with primary antibodies for both IB4 and Iba1 (which can label activated microglia) revealed clear co‐localization of staining in these regions. Spinal intact rats (N=5) had robust IB4 staining in lamina II, but staining was not observed in the corticospinal tract, or ventrolateral or ventromedial white matter. The IB4‐Iba1 co‐localization after Hx likely indicates neuroinflammation and degeneration of damaged tracts. We also observed that IB4 immunostaining broadly dispersed throughout white and gray matter, in a pattern consistent with blood vessel endothelium labeling. To verify this, tissues were co‐incubated with primary antibodies against IB4 and vWF, a marker of endothelial cells. Co‐localization of vWF and IB4 immunostaining was detected throughout the mid‐cervical cord, and IB4 staining intensity was decreased in ventral gray matter caudal to Hx (P<0.05 vs. spinal intact). Reduction of IB4 intensity is consistent with vascular damage and regional tissue hypoxia. We conclude that following chronic C2Hx in adult rats, immunostaining with IB4 can identify blood vessel endothelial cells and activated microglia.

Support or Funding Information

1R01HL139708‐01A1 (DDF), OT2 OD023854, T32‐HD043730 (MDS), K99 HL143207‐01 (KS).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Early life vincristine exposure does not prime the developing pain circuitry for subsequent injury
Authors: Styczynski Lauren , Baccei Mark
Published: June 2021

Our recent work has demonstrated that the early life administration of vincristine (VNC), commonly used to treat pediatric leukemias, evokes peripheral neuropathy and mechanical pain hypersensitivity in rats that persists into adolescence. However, whether neonatal VNC treatment alters baseline pain sensitivity throughout adulthood remains unknown. It is also unclear if pediatric VNC exposure can ‘prime’ developing nociceptive pathways, and thereby exacerbate chronic pain, following subsequent trauma later in life. Therefore, the present study investigated the degree to which neonatal VNC administration influences mechanical and thermal pain hypersensitivity in the absence or presence of tissue injury sustained during adulthood. Rats received five total doses of 60 mg/kg VNC (or vehicle) on postnatal days (P) 11, 13, 17, 19 and 21, which is known to evoke mechanical pain and skin denervation by P26 (Schappacher et al., 2017). Immunohistochemical analysis of the skin suggested that a resolution of the peripheral neuropathy occurs by 13–15 weeks of age, as there were no differences in the density of intraepidermal nerve fibers or activated Langerhans cells between the VNC and vehicle‐treated groups at this time point. Baseline mechanical pain sensitivity, as measured with von Frey hairs, was also similar between the experimental groups during adulthood, confirming recovery to neonatal VNC treatment. To explore the potential long‐term effects of early life VNC on the behavioral response to subsequent insults, a surgical incision of the hindpaw was administered to adult rats exposed to either VNC or vehicle during the neonatal period. We observed no significant overall effect of early life VNC on the magnitude of post‐operative pain following adult incision. In addition, in order to model the clinical scenario where cancer relapse necessitates a second round of chemotherapy, separate groups of rats that had been treated with VNC (or vehicle) as neonates were subsequently administered VNC during adulthood (five injections at 100 mg/kg). There was no significant effect of prior VNC exposure on the level of mechanical pain hypersensitivity produced by adult VNC treatment. Collectively, these findings suggest that neonatal VNC does not increase the susceptibility to develop chronic pain as adults.

Support or Funding Information

Supported by National Institutes of Health (NS080889 to MLB).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gut microbiome and its potential role in obesity‐induced neuropathy
Authors: BONOMO RAIZA , Cook Tyler , Gavini Chaitanya , Mansuy‐Aubert Virginie
Published: June 2021

Obesity is considered a 21st century epidemic, affecting over 2 billion people worldwide1. This condition is often accompanied by several complications, including peripheral neuropathy (PN). PN is a highly prevalent disease, characterized by a distal‐to proximal nerve degeneration with current no available treatment2. The initial stages of PN are clinically presented as peripheral tactile allodynia and thermal hyperalgesia, that later progress to numbness and loss of sensation3. Despite high prevalence, the molecular mechanisms underlying the disease onset are poorly understood, and affected patients have no choice other than to use pain killers. Using sensory neurons‐specific transgenic mice, we observed expression of short chain fatty acid receptors (FFAR) at the surface of neurons expressing Nav1.8 (sensory neurons proven to be involved in type II diabetes painful neuropathy4,5). Because, short chain fatty acids are metabolites secreted by gut bacteria, this data potentially links the microbiome with obesity‐induced pain. Numerous studies have demonstrated that the gut microbiome may play a role in the development of obesity and its associated comorbidities6. Moreover, fecal microbiome transplantation (FMT) has been used to treat innumerous metabolic disorders7. Still, the relationship between gut microbiome and PN onset and progression has yet to be explored. To investigate whether gut microbiome is involved in allodynia onset and/or progression in obesity, we utilized an obesity‐induced allodynia mouse model developed in our lab. Our results show that mice fed Western diet (WD) had reduced mechanical allodynia and thermal sensitivity threshold when compared to mice fed normal chow (NC). Our data showed that WD fed mice subjected to FMT, exhibited improved mechanical and thermal sensitivity when compared to WD alone and respective control groups. In addition, glucose tolerance was improved in the aforementioned groups. We also evaluated changes in i) gut bacterial composition following FMT, assessed by 16S sequencing, ii) in plasma short chain fatty acid and iii) in the gene expression in neurons of the dorsal root ganglia and sciatic nerve. In summary, the data presented shows that transplantation of gut bacteria from lean mice to WD‐fed mice improve PN symptoms in obese mice. Our data also suggest that the gut microbiome metabolites may change sensory neurons function via modulating short chain fatty acid receptor located at the surface of sensory neurons. This line of study could provide alternative therapeutic targets to replace the use of addictive pain killers. The gut microbiome, its metabolites and short chain fatty acid receptors could be a novel valuable target to study the delay or cure of debilitating pain associated with obesity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Exercise Pressor Reflex Evoked by Static Contraction is influenced by the Active Skeletal Muscle Mass in Decerebrated Rats
Authors: Estrada Juan A , Ducrocq Guillaume P , Kaufman Marc P
Published: June 2021

The decerebrated rat is a useful preparation to study the neural mechanisms controlling the circulation during exercise. One of these mechanisms, the exercise pressor reflex, is evoked by statically contracting the hindlimb muscles. However, contraction of only one hindlimb may not always be enough to achieve threshold for activation of the exercise pressor reflex in the decerebrated rat preparation. Here we demonstrate that simultaneous contraction of both hindlimbs evoked a greater pressor response than did contraction of one hindlimb. This was found to be the case when we analyzed the pressor response to contraction as either the area under the curve or the peak (n=4). Static contraction was induced by electrically stimulating the sciatic nerve for thirty seconds at currents that did not activate the axons of group III and IV afferents. When analyzing the area under the curve, we found that simultaneous contraction of both hindlimbs yielded a pressor response averaging 138 ± 64 mmHg seconds, whereas individual contraction of either hindlimb yielded a pressor response averaging either 48 ± 48 or 76 ± 23 mmHg seconds (right limb vs left limb, respectively). When analyzing the peak, simultaneous contraction of both hindlimbs yielded a pressor response averaging 12 ± 4 mmHg, whereas individual contraction of either hindlimb yielded a pressor response averaging 8 ± 6 mmHg or 9 ± 4 mmHg (right limb vs left limb, respectively). Additionally, the sum of the pressor responses to contraction analyzed as either the area under the curve or as the peak appeared to be additive. Our results suggest that the total active muscle mass influences the magnitude of the exercise pressor reflex.

Support or Funding Information

Supported by NIH grant P01 HL 134609.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exaggerated Pressor Response to Intermittent Contraction in Type 1 Diabetic Rats
Authors: Grotle Ann‐Katrin , Huo Yu , Ybarbo Kai M , Stone Audrey J.
Published: June 2021

Type 1 diabetes mellitus (T1DM) is associated with an impaired regulation of autonomic and cardiovascular responses during exercise, an effect attributable, in part, to the exercise pressor reflex. Previously, we have found that the pressor responses to both static muscle contraction and to tendon stretch are exaggerated in the early stage of T1DM. However, it is not known whether a similar response is present during dynamic exercise. Moreover, other studies have suggested that endothelial dysfunction manifests early in T1DM, which may impair muscle perfusion during exercise leading to an increased stimulation of the exercise pressor reflex. Therefore, the purpose of this study was to test whether T1DM rats have an exaggerated pressor and an attenuated blood flow response to intermittent contraction. To do this, we measured the exercise pressor reflex in unanesthetized, decerebrated T1DM (Streptozotocin 50mg/kg i.p; STZ: body weight: 328 ± 39g) and healthy (CTL: body weight: 264 ± 17g, n=5) Sprague Dawley rats by intermittently contracting the hindlimb for 30 s while continuously measuring mean arterial pressure (MAP), heart rate (HR) and popliteal arterial blood flow (BF). Popliteal vascular conductance was estimated by dividing popliteal BF by MAP. BF and VC are reported as mean change from baseline.

Muscle contraction significantly increased peak MAP (STZ: 32 ± 4 mmHg, n=3; CTL: 17 ± 4 mmHg, n=5, p<0.05) but not HR (STZ: 20 ± 4 mmHg, n=3; CTL: 14 ± 6 mmHg, n=5, p>0.05) to similar developed tensions (p>0.05) in T1DM rats compared to CTL rats. Muscle contraction increased hindlimb BF (STZ: 858 ± 379 μl/min, n=3; CTL: 220 ± 40 μl/min, n=2) and hindlimb vascular conductance (STZ: 7.4 ± 4.1 μl/min/mmHg, n=3; CTL: 2.2 ± 0.3 μl/min/mmHg, n=2) from baseline in STZ and CTL rats, however, this was not significantly different between groups (p>0.05). These findings are consistent with our previous studies that found that early stage T1DM rats had an exaggerated pressor response to static muscle contraction. Furthermore, the finding that blood flow delivery was not impaired in the T1DM rats may suggest that a mismatch between metabolic demand and oxygen delivery is not contributing to the exaggerated response.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nitrate increases in skeletal muscle peak power do not involve changes in sarcomeric protein phosphorylation and require whole‐body physiological pathways
Authors: Kumar Ravi , Hahn Dongwoo , Kelley Rachel , Ferreira Leonardo
Published: June 2021

Acute dietary nitrate supplementation in humans (within 2 hours) increases limb muscle peak power. In old mice, chronic (14 days) dietary nitrate supplementation increases diaphragm peak power. However, mechanisms of dietary nitrate effects on contractile mechanics are not fully understood. It is unclear whether nitrate ions directly or products downstream of the nitrate‐nitrite‐nitric oxide (NO) reductive pathway are responsible for changes in muscle contractile function. Increases in power likely involve post‐translational modification of myofibrillar proteins – phosphorylation being an established effect of NO signaling. We determined the phosphorylation status of myofibrillar proteins from diaphragms of 24 month old mice supplemented with nitrate (n = 8) or control (n = 6) and found no difference for myosin regulatory light chain, myosin binding protein c, tropomyosin, desmin or troponin I. Regulatory light chain phosphorylation signal normalized to total protein was 1.696 ± 0.139 and 1.699 ± 0.105 for control and nitrate‐supplemented animals, respectively. To examine a direct effect of nitrate on muscle cells, we exposed isolated diaphragm bundles from young mice in vitro to increasing concentrations of NaNO3. In vitro NaNO3 (15 min) had no effect on diaphragm peak power or isometric force (P > 0.05). Specifically, power (% control) was −1.06 ± 0.20 (0.5 mM), −1.73 ± 0.79 (1 mM), −2.64 ± 2.14 (2 mM), and −2.40 ± 2.40 (4 mM). Overall, our study shows that the increase in diaphragm power of aged mice elicited by chronic dietary nitrate supplementation does not involve changes in phosphorylation of key sarcomeric proteins that regulate contractile function. Moreover, our data from in vitro experiments suggest no direct effect of nitrate ions on mammalian skeletal muscle contractile function. Instead, dietary nitrate effects on muscle function appear to require whole‐body physiological mechanisms and actions of molecules downstream of nitrate.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.