bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cultivation of Hepatic Stellate Cells in 3D ECM to Simulate Physiological and Fibrotic Conditions
Authors: HAN YUAN‐PING 
Published: June 2021

BackgroundHepatic stellate cells, the major providers of hepatic retinoids and extracellular matrix (ECM), reside in the space of Disse filled with loose basement like ECM, and flanked by hepatocytes and sinusoidal endothelial cells. Upon hepatic injury, stellate cells are activated to express multiple matrix metalloproteinases (MMPs) and to mobilize retinoids for wound healing. In chronic injury, the quiescent stellate cells undergo trans‐differentiation into myofibroblast like cells, marked by loss of retinoids, expression interstitial ECM as well as smooth muscle actin (alpha‐SMA), and gain of contractibility. Although knowledge has been evolved substantially, two fundamental questions remain largely unknown as to how, in normal liver, HSCs maintain quiescence, and how, by injury, HSCs are activated within the ECM milieu.ResultsTo recapitulate the sub‐sinusoid microenvironment we embedded rat primary stellate cells in three‐dimensional ECM (3D ECM). Cultured on plastic, the stellate cells activated spontaneously, showing loss of vitamin A droplets and expression of alpha‐SMA. However, in 3D Matrigel, stellate cells retained retinoid droplets, in association with elevated level of lecithin retinoid acyltransferase (LRAT), a key enzyme for retinol esterification. In 3D ECM, stellate cells were arrested at G0/G1 phase, while on plastic the cells underwent cell cycling and stopped at G2/M phase. We then reconstituted the microenvironment of sinusoids by introducing Kupffer cells, the major hepatic inflammatory cells, into 3D ECM with HSCs. Activated Kupffer cells triggered subsequent activation of stellate cells, evidenced by expression of multiple MMPs, buildup of stress fibers, and loss of retinoids. The Kupffer cell‐mediated activation of stellate cells was blocked by interleukin‐1 receptor antagonist, demonstrating interleukin‐1 as a major injury signal from Kupffer cells conducting stellate cell activation and trans‐differentiation. Furthermore, within 3D ECM, but not on the plastic, interleukin‐1 signal was greatly amplified by stellate cells in an autocrine manner, which may exacerbate liver injury by expressing MMPs in the space of Disse. The myofibroblastic cells derived from the 3D ECM culture were contractile in response to endothelin.ConclusionThe 3D ECM culture provides a model to simulate key physiological functions of stellate cells in normal liver, and also to recapitulate patho‐physiological responses in liver injury. The method may also be used in cell‐based drug screening to identify components targeting liver failure and fibrosis.Support or Funding InformationNational Institutes of Health (NIH) Grants R01s, DK069418‐05 and AR051558, and the Natural Science Foundation of ChinaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Typing of Hepatitis B Using Fusion Primers and IonTorrent
Authors: Ringlander Johan , Andersson Maria , Lindh Magnus 
Published: June 2021

BackgroundHepatitis B virus (HBV) chronically infects 250 million people, and is the main contributor to hepatoceullular carcinoma (HCC) in the world. When contracted at adult age, HBV can cause acute hepatitis which sometimes is severe and may result in acute liver failure. Molecular epidemiology is important for the analysis of circulating HBV strains and for clarifying transmission chains. Next‐generation sequencing (NGS) has several advantages over traditional Sanger sequencing, but high costs for NGS, in particular the library preparation step, has restricted its use.ObjectiveTo develop a cost‐efficient strategy for simplified amplicon sequencing of HBV whole genomes on IonTorrent S5 (Thermofischer, Watlham, MA, USA), and an automated bioinformatics pipeline.MethodPrimers with tails carrying IonTorrent A or P1 adaptor, were designed to target conserved regions using an alignment consisting of fifty HBV strains, reflecting all known genotypes. The primers were located to cover the genome by 10 amplicons of approximately 380 bp size, and will be presented. HBV genomes in serum samples was amplified by duplex PCR in five parallel reactions, followed by sequencing in IonTorrent S5, and bioinformatic processing by CLC Genomics Workbench (Qiagen, Aarhus, Denmark).ResultsAll ten overlapping segments of HBV DNA from four patient sera, representing genotypes A, B, C and D were successfully amplified. Sequencing data are yet to be analyzed and will be presented.Discussion and conclusionTo our knowledge, whole genome sequencing of HBV using fusion primer derived amplicons for IonTorrent have not previously been published. By this technique, whole genome sequencing of HBV can be performed in large scale and at low cost.Support or Funding InformationGovernmental strategic research funds, Sahlgrenska University Hospital.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does Perfluorooctane Sulfonic Acid (PFOS) Affect the Mouse Immune System?
Authors: Torres Luisa , August Avery 
Published: June 2021

Perfluorinated alkyl acids (PFAAs) have been used in numerous industrial applications as stain and water repellants, floor waxes, fire‐fighting foams, denture cleaners, shampoos, carpet spot cleaners, pharmaceutical products, and pesticides. PFAAs contain simple carbon chains bound to fluorine atoms as well as various substitution groups on the terminal end that dictate their active properties. The strength of the carbon‐fluorine bond makes perfluorinated compounds extremely resistant to environmental degradation, thereby leading to persistence in the environment. Perfluorooctane sulfonic acid (PFOS) is the PFAA most commonly found in both human and wildlife blood samples. PFOS exposure has been linked to hepatotoxicity, neurotoxicity, endocrine disruption, and carcinogenicity, but little is known regarding its impact on the immune system. To assess the effect of PFOS on populations of immune cells, we exposed adult male and female C57BL/6 mice to a low dose of PFOS (5μg/kg/day) via oral gavage every other day for 12 or 30 days. We observed a reduction in the number and percentage of lymphocytes as well as a reduction in the numbers of T cells and natural killer cells in the thymus and fat tissue of the mice after 12 days of exposure. However, these changes were not observed at 30 days. These results suggest either a transient reduction in immune cells, or short‐term redistribution of immune cells to other tissues upon oral exposure to PFOS. Future work will determine the impact of longer‐term PFOS exposure on populations of immune cells, as well as effects on response to viral infections, and antibody responses.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glypican 3 (GPC3)‐CD81 axis regulates Ezrin mediated Hippo pathway via cross talking with HGF/c‐Met axis in hepatocytes and hepatocellular carcinoma (HCC)
Authors: Xue Yuhua , Mars Wendy , Bowen William , Orr Anne , Stoops John , Michalopoulos George 
Published: June 2021

The experiments that integrate different pathways in normal and abnormal circumstances will help us to develop optimum targeted therapies because of multiple pathways involved in the solid tumor carcinogenesis. We have shown that activation of CD81 is associated with phosphorylation of Ezrin, which is overexpressed in many types of solid tumors and has been identified as metastasis related protein for many years. We have also shown that GPC3 binds to the membrane tetraspanin CD81, which is one of the portals of entry of hepatitis C virus (HCV). Previous studies have shown that GPC3 is associated with inhibition of hepatocyte growth and liver regeneration by inhibiting CD81‐mediated p‐Ezrin/Hippo/Yap pathway. It also has been shown that HGF/Met axis which promotes growth in HCC can phosphorylate Ezrin as well by different kinases. Since we have demonstrated the predominant GPC+++/CD81− pattern in HCC, the purpose of this study is to explore the regulation of expression and phosphorylation of Ezrin in HCC. We have found that CD81 expression could be detected in membrane and organelle fraction in human hepatoma cell lines HepG2, Huh7 and Hep3B, but immunoprecipitation experiment suggests that CD81 from latter two human hepatoma cell lines has lost association with GPC3. Human hepatoma cell lines have dramatically up regulated Ezrin expression and Yap activity. We found that treatment of primary rat hepatocytes with CD81 agonist antibody or HGF led to higher levels of phosphorylated Ezrin and lower Hippo activity. Mice overexpressing GPC3 had decreased p‐Ezrin but increased Hippo activity. In mice with post‐natal Met knock out there was less Yap expression at baseline level. Rat hepatoma cell lines JM1 and JM2 had dramatically down regulated CD81 expression and Hippo activity, and up regulated p‐Ezrin. Treatment with 20 umol of Ezrin inhibitor led to significant cell death selectively in JM2 cells in vitro.ConclusionsBoth HGF/Met and CD81 mediate p‐Ezrin activity and enhance nuclear Yap levels. These pathways in normal hepatocytes are inhibited by GPC3 and low HGF/Met activity. In human hepatocytes, GPC3 inhibits the CD81‐mediated p‐Ezrin effect on Hippo pathway and cause a decrease in nuclear Yap. The different distribution pattern of HGF/Met and GPC3/CD81 between normal hepatocytes and HCC cells provides the chance to develop precise anti‐HCC therapies. And Ezrin is a promising target of anti‐HCC therapies including primary lesion and metastasis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic Profiling of Serum Reveals Significant Changes in Lipid and Amino Acid Metabolism in A Lipodystrophy Mouse Model
Authors: Zhou Yawen , Cheng Kenneth KY , Xu Aimin , Hoo Ruby LC 
Published: June 2021

ObjectiveAdipose tissue serves as not only an energy reservoir to store excess energy in form of triglyceride but also as an important endocrine organ producing adipokines such as leptin and adiponectin. Lipodystrophy, characterized by abnormal or degeneration of adipose tissue, leads to metabolic diseases including hypertriglyceridemia, insulin resistance, and hepatic steatosis. Lipotoxicity is known as a major driver for the metabolic dysregulation in lipodystrophy, however, which specific lipid species and/or other factors contributes to the disease progression is not well characterized. In this study, we employed untargeted metabolomics to identify small metabolites that are altered in a lipodystrophy mouse model.MethodsWe have recently generated the lipodystrophy mouse model by which adipocyte mouse double minute 2 homolog (Mdm2) is deleted by Cre‐loxP approach. Circulating metabolites were measured and compared in the serum of adipocyte‐specific Mdm2 knockout mice at 3‐week (Before lipodystrophy development) and 12‐week old (After lipodystrophy development) with age‐matched wild‐type mice, using an untargeted metabolomics profiling by LC‐MS. Data were explored using principal component analysis and statistical analysis.ResultsOur untargeted metabolomics profiling had successfully identified and quantified more than 300 circulating metabolites in the serum samples. The results promptly point towards a significant increase in metabolites groups including carnitine and amino acids in the lipodystrophy mouse mice. During the development of lipodystrophy, serum level of phosphatidylcholines species (PC) was significantly reduced whereas increased serum sphingolipid species (including sphingosine and sphinganine) were observed in Mdm2 knockout mice is positively associated with insulin resistance. The high rate of incomplete fatty acid oxidation and accumulation of acylcarnitine metabolites may indicate the damage of skeletal muscle due to an excessive fatty‐acid metabolites supply in the lipodystrophy mice.ConclusionTogether, these data show that metabolic diseases in lipodystrophy mice is associated with the alteration of metabolites supply to other tissues. Moreover, the utilization of lipodystrophy model in investigating the mechanisms of novel therapeutic targets of metabolic disease via maintaining normal adipose tissue functions was demonstrated.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intraperitoneal myriocin upregulates transforming growth factor b1 (tgf‐b1) expression in murine destabilization of the medial meniscus model of osteoarthritis—evidence for a role of ceramide species in cartilage homeostasis
Authors: Rose Brandon J , Goodman Michael A , Kooyman David L 
Published: June 2021

Ceramides are biologically active sphingolipids known to play a role in numerous physiological and pathological processes. In particular, ceramides are known mediators in insulin resistance, and are found in increased abundance in osteoarthritic synovial fluid. Given the comorbidity between osteoarthritis and insulin resistance, we hypothesized that ceramides mediate cartilage catabolism in murine models of osteoarthritis. Further, that downregulation of ceramide synthesis would prevent structural damage and normalize biomarkers associated with osteoarthritis ‐ tgf‐b1, HtrA‐1, Ddr‐2, and Mmp‐13. To test this hypothesis, we performed surgical destabilization of the medial meniscus on the right knee of 8‐week‐old mice, treating one group with the serine palmitoyltransferase inhibitor myriocin and the other with a vehicle. An untreated sham group served as the control. We found myriocin administration failed to protect the joint from surgically‐induced cartilage degradation, and that no statistically significant difference occurred in the biomarkers measured with the notable exception of tgf‐b1, which was significantly increased in myriocin‐treated mice. Our findings lead us to the novel conclusion that ceramide species play an important role in cartilage homeostasis, acting specifically through regulating expression of tgf‐b1.Support or Funding InformationBrigham Young University ORCA GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA‐17‐3p inhibits excessive post‐hypoxic autophagy and attenuates H9C2 cardiomyocytes reoxygenation injury via PTEN‐Akt‐mTOR signaling
Authors: He Yi , Cai Yin , Yan Dan , Zhang Dengwen , Chen Chen , Irwin Michael G. , Wang Sheng , Xia Zhengyuan 
Published: June 2021

BackgroundThe microRNA (miR)‐17~92 is one of the best‐characterized polycistronic miRNA clusters, which encodes six individual miRNAs and contributes to various cellular and biological processes. In particular, miR‐17‐3p (a passenger miRNA of miR‐17) has been shown to indirectly inhibit Phosphatase and tensin homolog (PTEN) and attenuate myocardial ischemia/reperfusion (I/R) injury, but the underneath mechanism is unclear. Inhibition of PTEN can upregulate Akt/mTOR signaling and therefore suppresses the excessive autophagy, which may be a mechanism that underlies myocardial I/R injury. We, thus, hypothesized that miR‐17‐3p may protect against hypoxia/reoxygenation (H/R) induced cell injury by inhibiting post‐hypoxic excessive autophagy in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.Methods and resultsIn rat H9C2 cardiomyocytes, H/R (6 hours hypoxia followed by 6 hours reoxygenation) significantly enhanced the expression of miR‐17‐3p (P <0.05 vs. Control), which was concomitant by increased lactic acid dehydrogenase (LDH) leakage (cell injury marker, P<0.05 vs. Control) and the levels of p62 and the ratio of LC3II/I (autophagy markers, P<0.05 vs. Control), suggesting that miR‐17‐3p may be involved in the pathogenesis of H/R induced autophagic cell death. To explore the potential role of miR‐17‐3p in H/R‐induced cell injury, H9C2 cells were transfected with the miR‐17‐3p agomir or its negative control. The overexpression of miR‐17‐3p can significantly attenuate H/R‐induced cell injury (reduced LDH level, P <0.05 vs. H/R) and inhibit H/R‐induced excessive autophagy (decreased levels of p62 and LC3II/I, P <0.05 vs. H/R). Given the important role of PTEN‐Akt‐mTOR signaling in autophagy, the effects of miR‐17‐3p on the key events in the PTEN‐Akt‐mTOR signaling cascade during H/R were examined. As anticipated, miR‐17‐3p overexpression significantly down‐regulated PTEN expression and up‐regulated the levels of phosphorylated Akt1 (Thr308) and mTOR (P<0.05 vs. H/R), indicating that the miR‐17‐3p may protect against excessive post‐hypoxic autophagic cell death in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.ConclusionUpregulating PTEN‐Akt‐mTOR axis and the subsequent inhibition of excessive autophagy may represent the major mechanism whereby miR‐17‐3p attenuates H/R injury in H9C2 cardiomyocytes.Support or Funding InformationStudied supported by RGC/GRF grants (17158616M, 17117217 M)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of autophagy in cardiomyocytes by calmodulin antagonists
Authors: Lopez Vanessa , Keomanivong Faithe , Wauson Eric , Tran Quang‐Kim 
Published: June 2021

Macroautophagy (hereafter referred to as autophagy) is a catabolic process that regulates important aspects of myocardial metabolism in which cytoplasmic contents including proteins, other macromolecules, and organelles are sequestered in double membrane vesicles and delivered to lysosomes for destruction. While numerous studies have indicated involvement of signaling via Ca2+ in autophagic flux, the precise mechanisms remain unknown. Calmodulin (CaM) is the ubiquitous transducer of intracellular Ca2+ signals. CaM binds to an estimated network of over 300 cellular proteins, yet is expressed insufficiently for its targets. We have begun to examine the role of CaM in autophagic flux in cardiomyocytes. In H9C2 cardiomyocytes, amino acid starvation for 2 hrs triggers autophagic flux, as assessed by the formation of LC3‐II and the decrease in p62 protein levels. Chloroquine dose‐dependently reduces starvation‐induced autophagic flux. Competitive binding assays using purified CaM and FRET‐based CaM biosensors demonstrate direct CaM binding by chloroquine. Consistently, W‐7 and calmidazolium, two other structurally distinct CaM antagonists, dose‐dependently inhibit starvation‐induced formation of autophagic flux. These data demonstrate a role for CaM in autophagic flux in cardiomyocytes.Support or Funding InformationAHA 15SDG25090279 Grant (to E Wauson) and NIH HL112184 grant (to Q‐K Tran)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Signaling Pathways Contributing to Central Kappa Opioid Diuresis
Authors: Ebert Kevin M , Kapusta Daniel R 
Published: June 2021

Central activation of alpha‐2 receptors and nociceptin opioid peptide receptors (NOP) produce marked diuresis in rats, which is mediated in part by a downstream G‐alpha z protein signaling pathway that inhibits antidiuretic hormone (ADH) secretion. These studies investigated whether central G‐alpha protein signaling pathways also contribute in producing the diuretic and/or antinatriuretic response to central administration of the kappa opioid agonist, U‐50488H. Sprague Dawley rats were implanted with an intracerebroventricular (i.c.v.) cannula and pretreated with vehicle or pertussis toxin (PTX), which inhibits the Gi/o family of proteins except for Gz. After 48‐hrs pretreatment, rats were instrumented with cannula in the femoral artery, vein, and bladder and infused i.v. with isotonic saline (55 μl/min). After recovery, blood pressure (BP), heart rate (HR), and urine output (V) were collected in conscious rats before (control) and for 90‐min after (10‐min collections) i.c.v. U‐50488H (n=6). In vehicle pretreated rats, i.c.v. U‐50488H produced a marked increase in V (max Δ, 101±8 μl/min) and decrease in urinary sodium excretion (UNaV; max Δ, 5±0.5 μeq/min). The diuretic response produced by U‐50488H was significantly (p<0.05) blunted by pretreatment with PTX (max Δ, 57±3 ul/min). In contrast, PTX did not alter the decrease in UNaV. U‐50488H did not alter BP or HR in either group. To explore which G‐alpha proteins may participate in the kappa opioid mediated diuresis, separate rats were pretreated i.c.v. (24‐hrs) with an oligodeoxynucleotide (ODN), which selectively targeted a specific G‐alpha protein. In these studies, pretreatment of groups of rats (n=6/group) with a single ODN for either Gi1, Gi2, Gi3, Go, or Gz, failed to blunt the maximal diuresis as compared to the increase in V produced by i.c.v. U‐50488H in scrambled ODN treated rats. These results demonstrate that central kappa opioids produce a pertussis toxin sensitive diuresis, but not antinatriuresis. This suggests that a combination of Gi/o proteins may be required to produce the aquaretic effects of kappa opioid agonists.Support or Funding InformationSupported by NIH P30GM106392 to DRKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sphingosine‐1‐phosphate induced vasoconstrictor responses decreased in cerebral arteries of endotoxemic rats
Authors: SOYDAN GURAY 
Published: June 2021

IntroductionSepsis is the leading cause of mortality in intensive care units. Vascular dysfunction is the most important cause of death in sepsis. There are some data in the literature for vascular hyporesponsiveness to miscellaneous vasopressors. However most of the data are about resistance mesenteric artery and aorta which is the biggest artery in the body. The responses of cerebral arteries to vasopressors were not investigated in vitro while the brain blood flows were investigated in sepsis.Sphingosine‐1‐phosphate (S1P) is a bioactive lipid mediator which has several physiological functions in cell growth, inflammation, endothelial barrier integrity and vascular tone. Serum S1P levels decrease in sepsis and this decrement is correlated with the severity of sepsis. S1P is a vasoconstrictor lipid mediator which has more vasoconstrictor responses in small sized vessels and brain arteries. Our aim is to study the vasoconstrictor responses of the brain and mesenteric arteries to S1P in sepsis.Material and Methods10 mg/kg lipopolysaccharide (serotype O55:B5) were given intraperitoneally to rats to make an animal model os sepsis. Rats were anesthetized with gaseous carbon dioxide and decapitated 20 hours after injection, Basilar, anterior cerebral, middle cerebral and 3rd branch of mesenteric arteries were mounted in a wire myograph. Arteries were stimulated with 124 nM K+‐PSS after 30 min stabilization. Concentration‐response curves for S1P were evaluated.ResultsHigh K+ responses did not change in brain and peripheral arteries. S1P‐induced vasoconstrictor responses decreased in anterior and prominently in middle cerebral arteries. S1P‐induced vasoconstrictor responses in basilar and mesenteric arteries did not change.ConclusionsThis is the first study that show the alterations of S1P‐induced vasoconstrictor responses of brain arteries in sepsis. The difference in the alteration of S1P responses of cerebral arteries may explain the alteration and redistribution of cerebral perfussion in sepsis.Support or Funding InformationThis study was funded by Hacettepe University Scientific Research Projects Coordination Unit (TSA‐2017‐12848).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.