bluuebunny/crossref_metadata_2025 huggingface dataset:
METABOLIC RATE AS A FUNCTION OF AGE IN BROWN NORWAY AND LONG‐EVANS RATS
Authors: Gordon Christopher J , Bishop Britton L
Published: June 2021

Brown Norway (BN) rats are commonly used in aging studies but relatively little is known on their metabolic rate (MR) as with aging. There is considerable disagreement in this field with some studies indicating a decrease and others showing an increase in MR with advanced age. Understanding how MR varies with age is critical for the development of pharmacokinetic models of environmental toxicants in aged animals. Male rats of the BN and Long‐Evans (LE) strain were housed unrestrained inside a direct calorimeter maintained at 22 °C for three hr while dry and evaporative heat loss were monitored. The sum of dry and evaporative heat loss was averaged over the final hour in the calorimeter to calculate MR (W/kg). MR was assessed in rats ranging from 1 to 23 months in BN rats (N=34) and 1 to 12 months in LE rats (N=18). MR of both strains was ~25 W/kg at 1 mo and then dropped rapidly with aging and growth. After reaching an age of 4 mo, MR of both strains reached an asymptote. As age increased beyond 4 mo, MR of the BN and LE strains was approximately 7 and 5 W/kg, respectively. There was no indication of an increase with MR as rats approached senescence. Body weight of the LE strain at maturity was approximately double that of the BN strain. While body weight has a marked effect on MR, there were strain specific differences in MR that were independent of body weight. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.
Fasting increases antioxidant enzymes and glutathione content in elephant seals
Authors: Vazquez‐Medina Jose Pablo , Choi Jinah , Forman Henry J , Crocker Daniel E , Ortiz Rudy M
Published: June 2021

Northern elephant seals (NES) are naturally exposed to prolonged fasting (2–3 mo) while weaning, molting or breeding. In terrestrial mammals, fasting increases reactive oxygen species (ROS) production and oxidative damage. Fasting in NES activates the renin‐angiotensin system and increases the expression of the ROS‐producing protein Nox4 without increasing local or systemic indices of oxidative damage. The mechanisms NES evolved to cope with fasting‐induced ROS production remain unexamined. To test our hypothesis that antioxidant enzymes and glutathione (GSH) increase with fasting in NES, muscle biopsies and blood samples were collected from early and late fasted NES pups (N= 17). Antioxidant enzymes (superoxide dismutases [SOD], catalase [CAT] and GSH peroxidase [GPx]) muscle protein expression, their muscle and red blood cell (RBC) activity levels, and muscle and RBC GSH content were analyzed. Fasting increased antioxidant enzymes expression (40–60%) as well as muscle and RBC SOD (6 ± 2 vs 12 ± 4 U/mg protein; 788 ± 16 vs 920 ± 24 U/mL), CAT (29 ± 4 vs 69 ± 15 U/mg protein; 5192 ± 753 vs 8185 ± 973 U/mL) and GPx activities (6 ± 1 vs 10 ± 0.5 U/mg protein; 430 ± 90 vs 935 ± 149 U/mL). Muscle (4 ± 1 vs 19 ± 4 nmol/mg protein) and RBC GSH (0.5 ± 0.09 vs 1.3 ± 0.3, μmol/g Hb) also increased with fasting suggesting that endogenous antioxidants contribute to NES tolerance to fasting‐induced ROS production. Moreover, the accumulation of antioxidants during the fast may prepare NES to cope with dive‐induced ROS production, since fasting precedes the beginning of their diving lifestyle. Funded by NIH HL091767.
The Avian Pancreatic Islets: A Comparison Across Species
Authors: Hostetter Kimberly Gean , Sweazea Karen L. , Braun Eldon J
Published: June 2021

The mammalian pancreas has a remarkable ability to maintain glucose homeostasis, through the production of the blood glucose lowering hormone insulin, and its counter‐regulatory hormone glucagon. The concept of avian diabetes arose in early studies of Minkowski in the late 1800's, which demonstrated that removal of the pancreas in domestic fowl and geese did not result in the development of diabetes. A species that deviates from this is horned owls which develop hyperglycemia and die following pancreas removal. Prior studies have demonstrated that the chicken pancreas contains more glucagon‐producing alpha cells compared to insulin‐producing beta cells. This is reflected in low blood levels of insulin in chickens. We hypothesize that species differences may exist in the expression patterns of pancreatic islet cells in birds allowing some to persist after pancreatectomy, whereas other birds perish as a result of hyperglycemia. In the present study, we employed immunohistochemistry to compare the density of alpha and beta cells in pancreatic tissue across the avian species. Studies were conducted on Mourning dove, Hooded Siskin, Collared Warbling Finch, and House Wren and we found that alpha cells were denser in the avian pancreas relative to beta cells. The study has been extended into other species such as Cinereous Conebill, Rufous Collared Sparrow, Amazilia Hummingbird, and Mountain Parakeet.
Resveratrol‐induced ATF3 expression is mediated by EGR‐1/KLF4 interaction
Authors: Whitlock Nichelle C , Bahn Jae Hoon , Baek Seung Joon
Published: June 2021

Resveratrol, a phytoalexin readily available in the diet, is reported to possess anticancer properties in colorectal cancer. However, the underlying mechanism involved is not completely understood. We investigated the affect of resveratrol on gene modulation in HCT116 cancer cells and identified activating transcription factor 3 (ATF3) as most highly induced. Resveratrol increased ATF3 expression at the mRNA and protein level. Promoter analysis revealed a major response element located within the −514 bp region in which four putative cis‐acting elements were located. Cotransfection of these factors with the −514 bp region of the ATF3 promoter pointed to the involvement of early growth response 1 (Egr‐1) and Kruppel like factor 4 (KLF4) in resveratrol induction of ATF3. Internal deletion of the Egr‐1 and KLF4 binding sites and suppression of their protein confirmed the importance of these cis‐acting elements. Specificity of these sites to the Egr‐1 and KLF4 protein was confirmed by EMSA and ChIP assays. Resveratrol increased Egr‐1 and KLF4 expression, which preceded ATF3, and further suggests their involvement in resveratrol mediated activity. Egr‐1 and KLF4 synergize to activate ATF3 and bind each other; both enhanced by resveratrol and may facilitate ATF3 transcriptional regulation by this compound. Taken together, these results provide a novel mechanism by which resveratrol induces ATF3 expression.
Thioamide derivative‐induced cytotoxic effect in human liver carcinoma (HepG2) cells
Authors: Young Jarrod , Yedjou Clement , Cameron Joseph A. , Tchounwou Paul
Published: June 2021

Thioamide is an antithyroid drug that has been shown to cross the placental barrier. Use of antithyroid medications in pregnancy may result in fetal hypothyroidism, goiter, and cretinism. The literature review indicated that about 10% of patients taking thioamide drug have skin eruptions, maculespapules, urticaria, dermatitis, fever, and arthralgia. However, the precise mechanisms involved in thioamide toxicity are not well elucidated. In the present study, we use human liver carcinoma (HepG2) cells as a test model to evaluate the cytotoxicity of three thioamide derivatives. To achieve this goal, cell survival was determined by the means of MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐ diphenyltetrazolium bromide, a tetrazole) assay. Human liver carcinoma (HepG2) cells were treated with different doses of thioamide derivative for 48 hours. Results from MTT assay indicate that thioamide derivative gradually reduce the viability of HepG2 cells in a dose‐dependent manner, showing LD50 value of about 13.58, 32, and 38μM, upon 48 h of exposure, respectively. Based on this in vitro, our thioamide derivative appears to be highly cytotoxic to HepG2 cells. This research was supported in part by NIH‐NIGMS Grant R 25 GM 50117, and in part by a grant from the National Institutes of Health, Grant No. 2G12RR013459‐11, through the RCMI‐Center for Environmental Health at Jackson State University.
Effect of YWK‐II/APLP2 on neuron survival
Authors: Sun Tingting , Chan Hsiao Chang
Published: June 2021

YWK‐II protein, the target antigen to a monoclonal antibody raised against human sperm proteins, has been recently implicated in sperm survival and anti‐apoptotic activity acting as a novel receptor for Mullerian inhibiting substance (MIS). Sequence of YWK‐II protein is highly homologous to amyloid‐precursor‐like protein 2 (APLP2) that belongs to a conserved transmembrane family including amyloid precursor protein (APP). In the present study, we investigated the potential role of YWK‐II protein in neuron survival. RT‐PCR and Western blot showed that YWK‐II is expressed in primary cultured hippocampal neuron and PC12 cells both at mRNA and protein levels, while both cells lack MIS expression. In serum withdrawal medium, viable hippocampal neuron and PC12 cells were significantly decreased as assessed by MTS assay, indicating possible cell death. Recombinant MIS (rMIS) could protect hippocampal neuron from serum‐withdrawal induced death, but it did not affect PC12. Addition of YWK‐II antibody could block the protective effect of rMIS. Moreover, the presence of YWK‐II antibody alone could reduce viable cells in both cells. The results of our study indicate the possible involvement of YWK‐II protein in neuron survival, which may be partially mediated via MIS in vivo.

The present study is funded by National 973 Program (2006CB504002).
Midkine and Hypoxia Potentiate Epithelial‐to‐Mesenchymal Transition in Human Lung Cancer Cells
Authors: Zhang Hanying , Zawada Michael , Das Mita
Published: June 2021

Epithelial‐to‐mesenchymal transition (EMT) plays an important role during progression of lung cancer. Recently, hypoxia and midkine (MK) have been shown to be key regulators of EMT in various types of cancer cells. Signal transducer and activator of transcription 3 (STAT3) also plays a critical role in this process. Therefore, we hypothesize that MK will enhance EMT in lung cancer cells grown in a hypoxic microenvironment through STAT3 activation. Human lung cancer cells (A549) were exposed to 1% oxygen in the presence of MK to test our hypothesis. EMT was evaluated by assessment of cell morphology as well as expression of marker proteins. Cell morphology was altered from cuboidal to elongated shape after 48 hours of treatments. Levels of E‐cadherin, marker of epithelial cells, were decreased. However, expression of alpha smooth muscle actin (αSMA) and vimentin, markers of mesenchymal cells, were augmented. Hypoxia/MK treatment stimulated STAT3 phosphorylation at Ser727 as well as Tyr705 residues. Interestingly, the two phosphospecies had unique subcellular localization patterns. PhophoSTAT3(Tyr705) was restricted to focal points within the cytoplasm, whereas Ser727 phosphoSTAT3 was present predominantly in the nuclear compartment. Therefore, our data strongly suggest that hypoxia/MK potentiates EMT process and this process might be mediated through STAT3 activation in human lung cancer cells.

Funded by HL64917 (MD); P20RR016474 (NCRR & Wyoming INBRE)
The Role of Lung Mesenchymal Stem Cells in the Progression of Pulmonary Fibrosis
Authors: Majka Susan M , Jun Du
Published: June 2021

Introduction

Pulmonary fibrosis (PF) is often complicated by pulmonary arterial hypertension (PAH) in adults. We hypothesize that during PF and PAH the mesenchymal stem cell (MSC) differentiation program that normally generates reparative cells is altered, leading to the generation of proliferative myofibroblasts, that contribute to vascular wall thickening.

Methods

To identify putative lung MSC, we performed flow cytometry on single cell lung suspensions to isolate Hoechstlow/CD45neg cells. We then characterized the MSC phenotypic and functional characteristics of this population. Next, we tested whether lung MSC could affect the pathophysiology of PF/PAH.

Results

Hoechstlow/CD45neg lung cells demonstrated multi‐lineage differentiation. We found that these cells, as mixed or clonal populations, have the capacity to differentiate into several mesenchymal cell lineages, EC and myofibroblasts. In addition, similar to bone marrow‐MSC, bleomycin induced PF and PAH was significantly attenuated in animals injected with lung MSC in the absence of detectable engraftment on day 14. However, lineage analyses using the novel SP marking system demonstrated the incorporation of endogenous MSC to fibrotic regions as well as vasculature on day 14 following bleomycin exposure.

Conclusions

Our results illustrate the presence of a lung MSC population and the importance of studying stem cells in the context of their niche to understand their true role in the progression of disease processes.
High salt intake exacerbates renal tissue oxidative stress and urinary angiotensinogen excretion during AngII‐dependent hypertension.
Authors: da Silva Lara Lucienne , McCormack Michael , Kobori Hiroyuki , Shenouda Sylvia , Majid Dewan SA , Navar Luis Gabriel , Prieto Minolfa Carrasquero
Published: June 2021

High salt (HS) intake inhibits systemic and intrarenal RAS; however, when AngII is not suppressed HS exacerbates hypertension and renal injury. We investigated the effects of HS on the magnitude of intrarenal oxidative stress associated with angiotensinogen urinary excretion (uAGT) in chronic AngII‐infused rats. In Sprague‐Dawley rats: 1) Control, normal salt diet [NS, n=5]; 2) HS diet [8% NaCl, 12 d, n=5]; 3) AngII‐infused in NS rats [AngII, 80 ng/min, 14 d, n=5] and 4) AngII‐infused in HS rats [AngII+HS, n=5] we measured systolic blood pressure (SBP), proteinuria, NADPH oxidase activity, n‐Tyr residues and uAGT. HS alone did not change SBP, but led to hypertension when combined with AngII. In contrast, HS alone led to proteinuria that was further increased in HS+AngII rats. The uAGT excretion rates increased in AngII+HS rats compared to AngII rats (AngII=107±18 vs AngII+HS=4363±1296 ng/day). Renal tissue oxidative stress measured by n‐Tyr Western blot increased in both HS and AngII+HS rats; however the NADPH oxidase activity increased only in HS+AngII rats (HS=10,674±2819; AngII+HS=18,663±1328 RLU/mg protein). HS exacerbates renal tissue oxidative stress during AngII‐dependent hypertension which might be associated with increased uAGT, proteinuria, and blood pressure in AngII‐infused rats.

NIH [IdeA Program (P20RR01765906); Tulane‐BIRCWH Program (K12HGO4351)] and AHA (09PRE2209079; 09BGIA2280440)
Glucagon‐Like Peptide (GLP1) Agonist Increases GFR and Suppresses Proximal Reabsorption
Authors: Thomson Scott Culver , Kashkouli Ali , Singh Prabhleen
Published: June 2021

The incretin, GLP1, is released by the gut in response to fat or CHO and lowers glucose by effects on the stomach and pancreas. GLP1 receptors are also in kidney. We examined the effects of a GLP1 agonist, exenatide, on glomerular and tubular function in normal and diabetic rats.

GFR (inulin clearance) and urine flow rate were measured in awake rats following exenatide (1 nmol sq) or placebo (n=5 each). Micropuncture was done in normal and hyperfiltering STZ‐diabetic rats before and during exenatide (1 nmol/h iv). Single nephron (SN) GFR and proximal reabsorption (Jprox) were measured at both extremes of tubuloglomerular feedback (TGF) activation (loop of Henle perfusion). ANCOVA with SNGFR as covariate tested for primary effects on Jprox.

Conscious rats on exenatide had higher GFR (4.3±0.2 vs 2.2±0.3 ml/min, p=0.001) and urine flow (34±4 vs 12±3 ul/min, p=0.002). Micropuncture results are shown in the table‐*P<0.05 for exenatide. Total 114 tubular fluid collections from 6 rats.

SNGFR no TGF (nl/min)
SNGFR max TGF
Jprox adjusted for SNGFR

Control
40±5
29±7
26±1

+ Exenatide
70±4*
50±6*
18±1*

Diabetes
52±5
33±6
26±1

+ Exenatide
65±4*
53±6*
16±1*

Exenatide increases GFR and is a potent proximal diuretic, implying effects on both glomerulus and tubule. It is a mystery why the GLP1 receptor, whose natural agonist responds to fat ingestion, should have such capacities to raise GFR and suppress proximal reabsorption.
Dopamine D2 receptor regulation of reactive oxygen species production via paraoxonase 2
Authors: Yang Yu , Zhang Yanrong , Villar Van Anthony , Pascua Annabelle , Armando Ines , Jose Pedro A.
Published: June 2021

An intact dopaminergic system is necessary to maintain normal blood pressure. We have reported that D2 dopamine receptor (D2R) regulates renal reactive oxygen species (ROS) production and that altered D2R function results in ROS‐dependent hypertension. Paraoxonase 2 (PON2) belongs to the paraoxonase gene family that is expressed in various tissues including the kidney and protects against cellular oxidative stress.

We hypothesized that PON2 may be involved in counter‐regulating excessive renal ROS production and thus may contribute to maintain normal blood pressure. Moreover, D2R may regulate ROS production in part through regulation of PON2. ROS production decreased (42%; P<0.05; n=4) in human renal proximal tubular (iNT) cells treated with the D2R agonist, quinpirole (24h, 1μM). This effect was associated with increased PON2 expression of mRNA and protein and enzymatic activity. PON‐2 silencing (siRNA, 10nM, 48h) down‐regulated PON2 expression (50%; n=3) and activity and increased ROS (34.9%, P<0.05). Furthermore, renal PON2 expression was down‐regulated in D2−/− mice (35.6%, P<0.05).

These results show that PON2 expression and/or function are closely related to ROS production and suggest that the D2R is involved in its regulation. The increased oxidative stress due to D2R disruption could be related to the failure of its stimulatory effect on the expression or function of PON2 in the kidney.

This work was supported in part by grants from the National Institutes of Health, HL68686, HL23081, HL074940, HL092196 and DK39308
The Largest Last Supper: How Depictions of Portion Size Have Increased Over the Millennium
Authors: Wansink Brian , Wansink Craig Steven
Published: June 2021

In recent years, public health awareness has shifted from food shortages and insecurity to the downsides of food abundance and obesity. If art imitates life, and if food resources have become generally more available over the past millennium, we might expect see its increased prominence to be reflected in paintings that depict food. Perhaps the most commonly painted meal has been the painting of Christ's Last Supper, chronicled in the New Testament of the Bible. A CAD‐CAM analysis of the relative size of food in 52 representative paintings of the Last Supper showed that the relative size of the main dish (r=0.52, P=.002), bread (r=0.30, P=.04) and plate (r=0.46, P=.02) have linearly increased over the past millennium.
Endothelin‐1 (ET‐1)‐induced reactive oxygen species (ROS) generation requires calcium (Ca2+) influx in pulmonary arterial smooth muscle cells (PASMCs)
Authors: Undem Clark , Luke Trevor , Sylvester J T , Shimoda Larissa A
Published: June 2021

ET‐1 is a potent endothelium‐derived vasoconstrictor and smooth muscle cell mitogen. During hypoxia, ET‐1 is thought to play a significant role in modulation pulmonary vascular tone. Previous studies in systemic smooth muscle have shown that both an increase in intracellular Ca2+ concentration ([Ca2+]i) and the generation of ROS via nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH oxidase) are major components of ET‐1 signaling. It remains unclear, however, whether ET‐1 causes ROS generation in PASMCs. In this study, we used the ROS‐sensitive dye, DCF, and the Ca2+‐sensitive dye, fura‐2, to determine 1) the effect of ET‐1 on ROS production in transiently cultured (24–48 hr) rat PASMCs and 2) whether this response is Ca2+‐dependent. Measuring both [Ca2+]i and ROS simultaneously, we found that ET‐1 significantly increased both Ca2+ influx and ROS generation, with Ca2+ influx appearing to occur first. Pre‐treating PASMCs with the antioxidant, TEMPOL, or the NADPH oxidase inhibitor, diphenyleneiodonium chloride, had no significant effect on ET‐1‐induced Ca2+ influx but prevented ROS production. In contrast, the removal of extracellular Ca2+ or pretreatment with the Ca2+ chelator, BAPTA‐AM, prevented both the increase in [Ca2+]i and ROS production. These results indicate that, in PASMCs, Ca2+ influx may be necessary for the ET‐1‐induced initiation of ROS production from NADPH oxidase.

HL67191
Rate constants suggest three mechanisms for porcine pulmonary arterial (PPA) contraction
Authors: Badenhop Sarah B , Desormeaux Victoria , Kumar Aswin , Conover Sam , Fawley Nick , Ha Soo , Hess Lauren , Pelaez Nancy
Published: June 2021

Vascular Smooth Muscle (VSM) uses both extracellular calcium ([Ca2+]o) and intracellular stores of calcium to signal a typical contraction. In contrast we find that fourth generation porcine pulmonary artery (PPA) contracts after high potassium depolarization with and without the presence of [Ca2+]o. The contractile behavior of PPA was modeled using JSIM Physiome Modeling software. PPA contraction in the presence of [Ca2+]o was characterized by both a fast and slow rate constant, whereas only a slow rate constant was observed without the presence of [Ca2+]o. Only this slow rate constant was detected in the presence of various calcium influx inhibitors including tetrandrine, caffeine and ethylene glycol tetraacetic acid (EGTA). High potassium‐induced contraction of PPA, with either the absence of [Ca2+]o or the inhibition of Ca2+ entry via voltage gated Ca2+ channels (VOCCs), suggests either the use of intracellular stores of Ca2+ through a novel signal transduction pathway or an unknown Ca2+ independent mechanism for contraction. PPA also contracted using H2O2, but with contractile rate that differed from those previously observed. The rates calculated from 3 mM H2O2 contractions were intermediate, but did not fit the exponential curve for the fast and slow rate constants. The intermediate curve was also dependent on [H2O2]. The intermediate rate constants suggest an alternative third mechanism underlying PPA contraction in response to H2O2.
Src‐family kinases mediate activation of RhoA and constriction in rat pulmonary artery
Authors: Shaifta Yasin , Snetkov Vladimir A , Aaronson Philip I , Ward Jeremy PT , Knock Greg A
Published: June 2021

The src family of tyrosine kinases (srcFK) are important upstream regulators of Rho‐kinase mediated Ca2+‐sensitization and constriction in rat pulmonary artery (RPA). We hypothesise that srcFK act through modulation of the activity of RhoA. Cytosolic to membrane translocation of RhoA in RPA smooth muscle cells, visualised using over‐expressed green fluorescent protein‐tagged RhoA was used as an indication of RhoA activation. Translocation was stimulated by prostaglandin‐F2α (20 μM), the superoxide generating quinone LY83583 (10 μM), and the RhoA activator calpeptin (0.02 U/ml). This translocation, following all three types of stimulus, was prevented by prior incubation with the srcFK antagonist PP2 (10 μM). Calpeptin also constricted myograph‐mounted RPA, and this constriction was inhibited by PP2. srcFK are likely to be activating RhoA through phosphorylation of one or more of the many proteins that directly activate or inhibit RhoA, namely guanine nucleotide exchange factors (RhoGEF), GTPase activating proteins (RhoGAP), and guanine nucleotide dissociation inhibitors (RhoGDI). We used RT‐PCR to confirm endogenous expression of p115‐RhoGEF, PDZ‐RhoGEF, LARG, Vsm‐RhoGEF, SmgGDS, vav‐3, p73‐RhoGAP, RhoGDI‐1 and RhoGDI‐2 in RPA. The GEF Vav‐2 was not expressed. Our data suggests that srcFK may be contributing to Ca2+‐sensitization and constriction of RPA through activation of RhoA.
Inflammation enhances Na+/Ca2+ exchange in human airway smooth muscle
Authors: Sathish Venkatachalem , Thompson Michael A , Pabelick Christina M , Prakash Y S , Sieck Gary C
Published: June 2021

Enhanced airway contractility by inflammatory cytokines involves increased intracellular Ca2+ levels in airway smooth muscle (ASM). In this study, we examined the role of the bidirectional plasma membrane Na+/Ca2+ exchanger (NCX) in human ASM Ca2+ regulation following exposure to the pro‐inflammatory cytokines tumor necrosis factor alpha (TNFα) or interleukin‐13 (IL‐13). Both cytokines significantly increased NCX1 expression, with these effects inhibited by dexamethasone (NFκB inhibitor) or PD98059 (MAPK inhibitor). In human ASM cells loaded with the Ca2+ indicator fura‐2, or the intracellular Na+ indicator SBFI, NCX‐mediated Ca2+ influx (with simultaneous Na+ efflux) was enhanced by both cytokines. NCX‐mediated Ca2+ efflux (with simultaneous Na+ influx) was also enhanced, but not to the same extent. The NCX inhibitor KBR7943 as well as NCX1 siRNA inhibited cytokine enhancement of both modes NCX and of Ca2+ responses to bronchoconstrictor agonists. These data indicate that a) NCX normally operates in human ASM; b) cytokines increase NCX expression, leading to greater NCX‐mediated Ca2+ influx, thus contributing to enhanced Ca2+ in inflamed ASM.

Supported by NIH grants HL74309 (GCS), HL090595‐S2 (VS) and HL090595 (CMP).
Arachidonate‐regulated channels (ARC) and intracellular Ca2+ regulation in human airway smooth muscle
Authors: Thompson Michael A , Pabelick Christina M , Prakash Y S
Published: June 2021

Plasma membrane Ca2+ influx and sarcoplasmic reticulum (SR) Ca2+ release are key components of intracellular Ca2+ regulation in airway smooth muscle (ASM). We and others have previously found that ASM Ca2+ regulation involves propagating Ca2+ oscillations via these mechanisms. Ca2+ oscillations in non‐excitable cells involve influx via arachidonic acid (AA) regulated channels (ARC); however, little is known about ARC in ASM. In human ASM cells loaded with fura2 (5 μM), we found that 10 μM AA triggered Ca2+ oscillations. OAG, oleic acid and palmitic acid (10 μM) failed to elicit Ca2+ responses. Pre‐incubation with LaCl3 1 μM and 1mM; 5 min) inhibited AA‐induced oscillations. Inhibition of SR Ca2+ release (ryanodine 10 μM and IP3 channel inhibitor Xestospongin C 10 μM; 30 min), decreased Ca2+ oscillation frequency and amplitude. Inhibitors of receptor‐operated channels (SKF‐96365; 10 μM), lipoxygenase (zileuton; 10 μM), or cyclooxygenase (indomethacin; 1 μM), did not affect oscillations. Finally, siRNA for Caveolin‐1, STIM1 and Orai3 (25nM) reduced frequency and amplitude of AA‐induced oscillations. These results indicate a novel caveolar Ca2+ influx mechanism in human ASM mediated via AA that interacts with SR to produce repetitive SR Ca2+ release. Such AA‐mediated influx could potentially contribute to increased Ca2+ in ASM during inflammation.

Supported by NIH grants HL090595 (CMP) and HL088029 (YSP)
Investigating alveolar mechanics and collapse via spherical indentation and in‐situ sub‐surface observation
Authors: Silva Maricris R , Shen Hsuan‐Tso , Gouldstone Andrew
Published: June 2021

Understanding of alveolar mechanics is important for respiratory physiology, pathology, and clinical anesthesia. In this study, we performed spherical indentation tests on excised mammalian lung lobes to explore alveolar stiffness and collapse. The experimental apparatus allowed force‐displacement data, and in‐situ microscopic observation of alveolar collapse and recovery under quantified applied stresses. Lungs were subjected to inhalation environments of air, 100% oxygen, and small amounts (0.2%) Isoflurane (IsoF), in different orders. Salient results of our studies include the following: 1) Inhalation of oxygen immediately increases lung stiffness and alters alveolar collapse distribution significantly; 2) Re‐opening of collapsed alveoli is strongly dependent on inhaled environment; 3) When the lung was inflated with air, then oxygen (3 tidal volumes) then air, the apparent effects of oxygen on lung mechanical properties were highly reversible. This data, coupled with preliminary experiments on isolated pulmonary surfactant under different environments, support our hypothesis that inhaled gases significantly, immediately and (sometimes) reversibly affect surfactant properties, and provide further evidence that damage associated with oxygen inhalation may be importantly associated with surfactant disruption.
Activity patterns of the thyropharyngeus (ThPh) muscle during eupnea, cough, and swallow
Authors: Pitts Teresa E , Rose Melanie , Poliacek Ivan , Sapienza Christine , Davenport Paul , Bolser Donald
Published: June 2021

Coordination of cough, breathing, and swallow is vital for airway protection. The role of the pharynx in coordination of these behaviors is not well understood. We speculated the ThPh would alter its activity to coordinate pharyngeal function during eupnea, cough, and swallow. Electromyograms (EMG) of geniohyoid (GH), ThPh, thyroarytenoid (TA), abdominal, and parasternal muscles were recorded in anesthetized cats (n=6). Coughing was produced by mechanical stimulation of the trachea and swallow was elicited by a water bolus in the pharynx. The ThPh EMG was expiratory modulated during eupnea. The ThPh muscle EMG amplitude increased in phase with the GH and TA EMGs during swallow. During tracheal stimulation, the ThPh EMG was suppressed, even if cough was not elicited. When swallows were induced during repetitive coughing, the ThPh muscle EMG burst behavior was similar to that during swallow alone. The ThPh is one of several pharyngeal muscles that control the aperature of the piriform sinus. Our results support modulation of the piriform sinus by tracheal afferents. This effect could promote pooling of material ejected by coughing in the piriform sinus in preparation for its removal from the pharynx by swallowing. The results support more complex coordinating mechanisms between airway protective behaviors than previously appreciated. Supported by NIH R33 89104.
Activity patterns of neurons in the caudal medial medulla are modulated during swallow in the cat
Authors: Rose Melanie J , Pitts Teresa E , Poliacek Ivan , Davenport Paul W , Morris Kendall F , Bolser Donald C
Published: June 2021

Neurons in the medial reticular formation, caudal to obex, have activity patterns that are modulated during cough and participate in assemblies with raphe and ventral respiratory column neurons. We speculated that neurons in this area would also participate in swallow. Extracellular activity of neurons in the caudal medial medulla was recorded with multiple electrode arrays in 5 spontaneously breathing, anesthetized cats. Electromyograms were recorded from the geniohyoid, thyropharyngeus, thyroarytenoid, posterior criciarytenoid, upper esophageal sphincter, transversus abdominis, and parasternal muscles as indicators of swallow and other airway protective behaviors. Swallow was produced by mechanical stimulation of the pharynx or infusion of water into this region of the airway. Less than 10% of the neurons were modulated during breathing. The activities of 54% of neurons were altered during swallow. Response patterns included increases or decreases in discharge of spontaneously active neurons and recruitment of silent neurons. In some cases, neurons also were excited by mechanical stimulation of the trachea. The results are consistent with a swallow medullary network that is more widespread than previously appreciated. Neurons in this area also participate in other airway protective behaviors, such as cough. Supported by NIH R33 89104.
Neural pathways that influence diaphragm activity and project to the lumbar spinal cord in cats
Authors: Rice Cory D , Weber Sarah A , Waggoner Allison L , Jessell Mary E , Yates Bill J
Published: June 2021

During breathing, the diaphragm and abdominal muscles contract out of phase. However, during other behaviors (including vomiting and postural adjustments) simultaneous increases in the activity of the diaphragm and abdominal muscles are required. Recent studies in cats using transneuronal tracing techniques showed that in addition to neurons in the respiratory groups, cells in the inferior and lateral vestibular nuclei (VN) and medial medullary reticular formation (MRF) influence diaphragm activity. The goal of the present study was to determine if neurons in these regions have collateralized projections to diaphragm motoneurons and the upper lumbar spinal cord, where abdominal motoneurons are located. For this purpose, the transneuronal tracer rabies virus was injected into the diaphragm and the monosynaptic retrograde tracer Fluoro‐Gold (FG) was injected into the L1 spinal segment. A large fraction of the neurons in the MRF and VN (median of 75 and 91%, respectively) that were infected by rabies virus were dual‐labeled by FG. These data show that MRF and VN neurons have connectivity with both phrenic and abdominal motoneurons, and thus can simultaneously regulate the activity of both the diaphragm and abdominal musculature. Future studies should focus on the physiological role of these neurons in producing co‐contractions of inspiratory and expiratory muscles.
Galanin (GAL)‐immunoreactive (ir) axons closely appose parvalbumin (Parv)‐immunoreactive neurons in the rat ventral respiratory column (VRC)
Authors: Llewellyn‐Smith Ida J. , Jung Kyoung L. , McCrimmon Donald R. , Alheid George F.
Published: June 2021

The neurochemistry of VRC neurons correlates with their function. Two‐thirds of large bulbospinal VRC neurons contain Parv and ~40% of chemosensitive neurons in the retrotrapezoid nucleus (RTN) contain GAL, which affects breathing when injected into the VRC. Here, we investigated whether GAL‐ir axons innervate Parv‐ir VRC neurons using double immunoperoxidase staining. In the VRC, GAL‐ir axons (black) closely apposed large Parv‐ir cell bodies (brown). We also identified two groups of black GAL‐ir somata. A rostral group occurred medial and ventral to the caudal end of the facial nucleus and are likely RTN neurons. A caudal group lay immediately ventral and slightly lateral to the column of Parv neurons, a location suggesting that some PreBötzinger (PreBöt) neurons might also be GAL‐ir. Since many PreBöt neurons contain the neurokinin‐1 receptor (NK1R), we double stained for GAL (brown) and NK1R (black). Some of the caudal GAL‐ir neurons were also NK1‐ir. These data suggest that GAL may influence respiration via PreBöt and/or RTN neurons releasing GAL onto Parv‐ir VRC neurons, which innervate spinal motor neurons that control respiratory pump muscles.

Support: NH&MRC of Australia & NIH grant HL088580.
Prediction of murine liver kinetics from plasma lipoprotein distributions
Authors: Tiemann Christian , van den Berg Sjoerd , van Klinken Jan Bert , van Dijk Ko Willems , Jeneson Jeroen , Hilbers Peter , van Riel Natal
Published: June 2021

The role of liver in lipid metabolism has been studied extensively. However, due to the complexity of in vivo liver measurements, many experimental efforts have focused on studying liver responses by measuring plasma lipoprotein distributions. The characteristics of these distributions co‐determine several metabolic disease risks and depend on production, clearance and remodeling of lipoproteins, all regulated by the liver. A challenge remains to determine the kinetics and distribution of fluxes in the complex network of metabolic pathways in the liver that give rise to these distributions. Here, we report on a computational approach to obtain this information.

A computational multi‐compartment model was constructed including reactions representing lipogenesis, cholesterol synthesis, fatty acid handling and lipoprotein metabolism in liver and lipoprotein remodeling in plasma. We consider a high‐resolution grid of different lipoprotein classes, from which lipoprotein distributions are calculated. Novel in this approach is the inclusion of lipoprotein kinetics, yielding additional information to constrain internal liver predictions and vice versa. In future, the model will be used to study the dynamic interactive behavior of plasma lipoprotein metabolism and metabolic pathways in murine liver for different physiological conditions. Funded by the Netherlands Consortium for Systems Biology.
Immunoparalysis: a new approach for its early detection and resolution
Authors: Torbati Dan , Lin Wei‐Chiang , Raszynski Andre , Totapally Balagangadhar R.
Published: June 2021

Persistence of the anti‐inflammatory reactions leads to immunoparalysis, characterized by reduced expression of pro‐inflammatory cytokines and the human leukocyte antigen‐DR in the blood. Reversal of immunoparalysis, in critically ill patients, could be life saving. Currently, cytofluorometric methods are used to detect changes in leukocytes functions, while the enzyme‐linked immunosorbent assay is used to measure the concentration of pro‐ and anti‐inflammatory cytokines. We propose to quantify the cumulative impact of the multiple inflammatory mediators, which are involved in development of immunoparalysis, by quantitative real time polymerase chain reaction. A simultaneous profiling of the pro‐ and anti‐inflammatory mediators at the mRNA level, combined with appropriate statistical analyses, may result in early detection of immunoparalysis. Statistically, a non‐parametric ANOVA could be used to compare expression scores of multiple mediators based on “fold change”, or the ratios of pro‐ and anti‐inflammatory mediators. Such ratios could be also evaluated by simple or complex chi square test if categorized as either opposing, or promoting immunoparalysis. The statistical outcomes could be used as indices for the onset/resolution of immunoparalysis in individuals, or a cohort of patients in clinical trials. The sensitivity and specificity of this approach warrants investigation in septic, and/or trauma patients.
Study of time‐dependent characteristics of a syllectrogram in the presence of aggregation inhibition
Authors: Namgung Bumseok , Ong Peng Kai , Jain Swati , Lim Dohyung , Chun Keyoung Jin , Kim Sangho
Published: June 2021

Erythrocyte aggregation is an important risk indicator of cardiovascular diseases, which can be quantified by dynamical parameters derived from a time‐dependent light transmission profile (syllectogram). This study tests the hypothesis that inhibitory factors on the aggregation process of the blood could greatly alter the dynamical information provided by the syllectogram and highlights the possible limitations in utilizing time‐dependent photometric methods for quantifying aggregation under such condition. The concept of the aggregation index (AIwin) that has been used in conventional aggregometers to determine extent of aggregation within a 12‐s standard time period was distinguished from the aggregation index (AI) derived from the aggregating phase of syllectogram. Apparent time delays in shape recovery and aggregating phases were observed at high levels of aggregation inhibition which accounted for the discrepancy between AI and AIwin. In the presence of a small shear, this discrepancy between AI and AIwin diminished. Fractal analysis was used to validate the aggregation results obtained from the syllectogram. AI was found to better depict the extent of red blood cell aggregation as compared to AIwin.
Rapid and Direct Quantitation of Gas Content in Liquids, Emulsions, and Blood
Authors: Rodriguez Juan , Newell Clayton , Simpkins Cuthbert
Published: June 2021

Here we describe new methodology for the rapid measurement of gas content in fluids, including simple liquids, emulsions, and blood. Unlike blood gas analyzers, our experimental approach measures gas content directly, i.e. without inferring it from the partial pressure of the gases in the aqueous phase. Our method incorporates a mass spectrometer detector that provides molecular specificity for O2, CO2, or other gases, so they may be detected by themselves or simultaneously. Mass spectrometry also affords ultra‐sensitive detection, which enabled us to measure O2 and CO2 content from 20 μL of whole blood with signal to noise ratios exceeding 1000:1. The ability to quantify gas content in microliter samples should enable the use of small animals for studying gas transport in plasma, whole blood, or even blood mixed with blood substitutes.
A human breathing lung‐on‐a‐chip
Authors: Huh Dongeun , Mammoto Akiko , Montoya‐Zavala Martin , Ingber Donald E.
Published: June 2021

Here we describe a biomimetic microsystem that reconstitutes the critical functional alveolar‐capillary interface of the human lung and exposes it to cyclic mechanical strain and fluid dynamic forces that mimic breathing and blood flow. This microdevice reproduces complex integrated organ‐level responses to bacteria and inflammatory cytokines introduced into the alveolar space by inducing expression of intercellular adhesion molecule‐1 (ICAM‐1) on the microvascular endothelium surface, adhesion of circulating blood‐borne neutrophils, their transmigration across the capillary‐alveolar interface, and phagocytosis of the infectious pathogens, which can be visualized using real‐time, high‐resolution microscopy. Using this approach, we developed novel nanotoxicology models and revealed that physiological cyclic mechanical strain greatly accentuates toxic and inflammatory responses of the lung to silica nanoparticles by promoting rapid release of toxic reactive oxygen species by alveolar epithelial cells and upregulating endothelial ICAM‐1 expression. Mechanical strain also enhances nanoparticle uptake by the epithelial cells and stimulates their transport into the underlying microvasculature. This mechanically active biomimetic microsystem represents valuable new model systems for in vitro analysis of various physiological functions and disease processes, in addition to providing low‐cost alternatives to animal and clinical studies for drug screening and toxicology applications. This work was supported by grants from NIH and the Wyss Institute for Biologically Inspired Engineering at Harvard University.
Equilibrium analysis between the venous return surface and cardiac output curve enables us to predict hemodynamic impact of percutaneous cardiopulmonary support (PCPS)
Authors: Tanaka Atsushi , Sunagawa Kenji
Published: June 2021

Background

PCPS is a vital tool in managing cardiogenic shock with respiratory failure. It remains difficult, however, to quantitatively predict the hemodynamic impact of PCPS. Theoretical analysis: We developed a circulatory equilibrium model in which cardiac output (CO) and venous pressures (right and left atrial pressures(PRA and PLA)) are determined as the intersection between the venous return (VR) surface and integrated CO curve. PCPS shifts the VR surface downward to the same extent of PCPS assisted flow (ΔCO). In contrast, PCPS increases left ventricular afterload pressure by R*ΔCO (R: arterial resistance) independent of ventricular ejection. Thus, PCPS lowers the equilibrium pulmonary CO and venous pressures. Using this model, we predicted CO and venous pressures under various levels of PCPS.

Methods and Results

In 5 anesthetized dogs (15–20 kg), we inserted PCPS and isolated carotid sinuses with bilateral vagotomy. We measured hemodynamics (CO, PLA, PRA, and aortic pressure) under various levels of PCPS support. Predicted COs and PLA correlated well with those measured (y=0.91x+6.9, r2=0.91, n=17, y=1.03x‐0.5, r2=0.93, n=17), whereas marginally correlated in PRA (y=0.69x+1.08, r2=0.73, n=17).

Conclusion

We could predict the impact of PCPS on CO and venous pressures. The proposed framework would be useful in managing patients with severely compromised hemodynamics.
Effects of Hydrostatic Pressure on Endothelial Tubulogenic Processes
Authors: Underwood Ryan Matthew , Shin Hainsworth
Published: June 2021

Fluid pressure stimulates endothelial cell (EC) elongation, cytoskeletal reorganization, and proliferation with the concomitant release of select growth factors such as basic fibroblast growth factor (bFGF). Since these responses are associated with capillary‐like tube formation related to angiogenesis, we hypothesize that pressure is a modulator of endothelial tubulogenesis. To address this, we used a custom‐designed pressure system to expose in two‐ or three‐dimensional cultures to sustained pressures of 40 mmHg in a standard tissue culture environment. On two‐dimensional substrates, bovine aortic endothelial cells (BAEC) exhibited elongated morphologies and increased cell densities after exposure to pressure for 48 hourrs. Moreover, results from our ongoing study show that BAEC cultured on Cytodex3© microcarrier beads in collagen gels and exposed to pressure in both the absence and presence of bFGF exhibited increased EC sprout and tube‐like structure formation. Our results, therefore, suggest that pressure influences endothelial tubulogenic processes. These findings provide a first step towards identifying conditions that promote optimal vascularization of tissue engineered constructs grown in vitro and for establishing an in vitro model to assess the role of pressure in physiological or pathological angiogenesis. Supported by the University of Kentucky Research Support Fund.
Simulated Repetitive Coughing at Vertical Tracheal Angles Significantly Improve Clearance of Simulated Gel‐like Mucus found in Patients with Respiratory Diseases
Authors: Ragavan Anpalaki Jeyabalasinkham , Evrensel Cahit A , Krumpe Peter
Published: June 2021

Interactive role of repetitive simulated coughing and vertical angles of trachea on clearance of gel‐like mucus produced by patients with respiratory diseases is analyzed in this study. Repetitive coughs (3 coughs of 0.1 sec. and 4 coughs of 0.075 sec. duration) were generated in a model trachea slightly larger in dimensions than average adult trachea placed at 4 vertical angles (0o, 15o, 30o, 45o), using a simulated cough machine. Mucus simulants with varying apparent viscosities (0.1 Pascal sec. (PaS) to 3 PaS) at relatively large storage modulus (13±1.0 Pa) were prepared to represent tracheal mucus of patients with various respiratory diseases. Rheological properties (storage mucudus (G') and apparent viscosity (η')) and displacement of leading and trailing edges of 0.3 ml simulant aliquots during coughs were measured. Repetition of coughs induced increased simulant clearance (p<0.05) compared to single coughs (0.3 sec duration) of the same velocity. The percentage increase in clearance was significantly larger at increased angles of the tracheal model (p<0.05). Significant negative correlations (−0.54) existed between percentage increase of clearance and η'. Results suggest that repetitive cough may increase the clearance efficiency of mucus with shear dependent properties compared to single cough of the same velocity and duration.
Are there changes in DNA methylation at tumor suppressor, imprinting or oncogenes in response to chronic consumption and withdrawal of folic acid?
Authors: Crider Krista Stimson , Bean Christopher , Berry Robert J , Rasmussen Sonja , Yang Thomas , Hao Ling , Zhu Li , Zhu Jainghui , Maneval David , Quinlivan Eoin , Bailey Lynn , Brant Jason
Published: June 2021

We evaluated the effect of folic acid supplementation and withdrawal on DNA methylation at 1505 loci in 802 genes using bead array technology. Blood samples from 95 Chinese women (stratified by three methylenetetrahydrofolate reductase [MTHFR] genotypes) participating in a double‐blind randomized trial of folic acid supplementation (100, 400, 4000 μg/d) were screened for changes in DNA methylation level and pattern at five time points: baseline, 1, 3 and 6 months supplementation and after a 3 month withdrawal. The overall mean baseline methylation patterns were highly correlated among the three MTHFR genotypes (r2 > 0.99). At baseline greater than 99.4% of these loci had variation in mean betas (ratio of methylated/unmethylated) of less than ±0.05 differences between the three MTHFR genotypes. After supplementation the variation among individuals in locus‐specific DNA methylation levels increased compared to the variation at baseline. Analysis is underway to determine if these changes are dependent on MTHFR genotype and time. Pyrosequencing will be completed to confirm any changes in loci detected through this screen. We did not find widespread DNA methylation changes associated with folic acid supplementation however, follow‐up of a limited number of changes is underway. This work was supported by a collaborative CDC agreement and the GCRC Grant # MO1‐RR00082.