bluuebunny/crossref_metadata_2025_split huggingface dataset:
Signaling Pathway by which β‐adrenoceptor Agonists Enhance Vascular Endothelial Growth Factor Release in Activated Human Macrophages
Authors: Ezeamuzie Charles I. , El‐Zohairy Sara , Oriowo Mabayoje 
Published: November 2023

Beta‐adrenoceptor agonists such as salbutamol are valuable bronchodilators, but they can also enhance the release of vascular endothelial growth factor (VEGF), which is capable of inducing tissue remodeling and support inflammation. In this study, we have characterized the signaling pathway involved in the enhancement of VEGF release by β‐adrenoceptor agonists in human macrophages. Human U937 cells differentiated into macrophages were primed with LPS in the absence or presence of β‐adrenoceptor agonists and antagonists. The VEGF released and the intracellular cAMP generated were assayed by ELISA. Isoprenaline, procaterol and salbutamol concentration‐dependently enhanced the release of VEGF induced by LPS in U937 macrophages. They also increased intracellular cAMP levels in these cells. BRL 37344, a selective β3‐adrenoceptor agonist, did not enhance VEGF release. Propranolol, ICI 118551 and atenolol all produced a parallel rightward shift of the isoprenaline dose‐response curve. The −logKB values were 8.12 ± 0.17, 8.03 ± 0.05 and 7.23 ± 0.05, respective, respectively, indicating clear involvement of both β1‐ and β2‐adrenoceptor subtypes. Activators of PKA, but not Epac system, enhanced VEGF release, and this effect was abolished by KT 5720 and Rp‐cAMPS ‐ both selective PKA inhibitors. These results show that β1‐ and β2‐, but not β3‐adrenoceptors, mediate cAMP‐dependent enhancement of VEGF release in LPS‐primed differentiated human U937 macrophages, and that PKA, not Epac, was the downstream effector of cAMP activity.Support or Funding InformationThis study was funded by Kuwait University Research Sector Grant # YM 14/09.
Investigating a Novel GPCR ITIM Motif in Regulating Shp2 Binding, Signaling, and Migration
Authors: Paulukinas Ryan D. , Belcastro Lili T. , Moore Catherine C. 
Published: November 2023

Aberrant expression of CXCR4, a chemokine G protein‐coupled receptor (GPCR), promotes a migratory and invasive phenotype during breast cancer metastasis, however the mechanism is unknown. Here we investigate the role of CXCR4 tyrosine phosphorylation within a novel ITIM motif. Following activation, CXCR4 is normally phosphorylated on Ser/Thr residues, which promotes receptor desensitization and internalization. We found previously that Ser/Thr phosphorylation of CXCR4 is delayed upon SDF gradient sensing, and correlates with sustained signaling to SHP2, a protein that drives hyperproliferation and invasion of breast cancer. Since SHP2 is known to be recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine‐based inhibitory consensus motifs), we investigated if CXCR4 contains an ITIM motif, and if tyrosine phosphorylation within this motif regulates SHP2 binding, signaling, and migration. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated as measured by recombinant phosphatase assay, phospho‐Ab development, and tyrosine mutagenesis (YF); 2) if tyrosine mutagenesis alters CXCR4 membrane localization, internalization, and SDF gradient sensing, by ELISA; 3) if tyrosine mutagenesis disrupts CXCR4 interaction with SHP2, by co‐IP; 4) if tyrosine mutagenesis alters CXCR4 sustained signaling to SHP2, by Western; and 5) if tyrosine mutation (YF) within the novel ITIM motif in CXCR4 alters migration of metastatic breast cancer cells, by transwell motility assays. Our results demonstrate that CXCR4 phosphorylation is sensitive to recombinant tyrosine phosphatase treatment and tyrosine mutagenesis, suggesting that CXCR4 is indeed tyrosine phosphorylated within the ITIM motif. We determined that while tyrosine phosphorylation within the ITIM motif is not required for CXCR4 membrane localization, internalization, or SDF gradient sensing, it does regulate CXCR4 binding and signaling to SHP2. Investigating the role in CXCR4 mediated migration is on going. To date, our data support a working model that tyrosine phosphorylation of CXCR4 within an ITIM motif is critical for binding to SHP2 and transducing sustained signaling to SHP2. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2.Support or Funding InformationThese studies were supported by NIH grant GM‐097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.
(−)‐Epicatechin Stimulates Mitochondrial Biogenesis in C2C12 Myotubes: Involvement of the G‐protein Coupled Estrogen Receptor (GPER)
Authors: Moreno‐Ulloa Aldo , Licea‐Navarro Alexei , Miranda‐Cervantes Adriana , Beltran‐Partida Ernesto , Donis‐Maturano Luis , De León‐Nava Marco , Villarreal Francisco , Álvarez‐Delgado Carolina 
Published: November 2023

During the last decade, it has been of great interest to identify natural products with promising effects on skeletal muscle function. In part, this has been fueled by the interest of developing small molecules with the capability to mimic and/or enhance the effects of exercise in order to improve muscle performance and more importantly to mitigate the effects of aging and disease‐induced muscle wasting. In this regard, our group has provided amply evidence as to how a natural small molecule that belongs to the family of flavonoids (subclass of flavanols), (−)‐epicatechin ((−)‐EPI), is capable to augment exercise capacity in mice and humans associated to an increase in mitochondrial function and biogenesis. The aim of this work was to evaluate the role that the GPER has on mediating the effects of (−)‐EPI on mitochondrial biogenesis in skeletal muscle cells. As an in vitro model of skeletal muscle cells we employed C2C12 myotubes. Using confocal microscopy we demonstrated that the GPER is localized in the cell surface and cytoplasm in C2C12 myotubes. The binding of (−)‐EPI to GPER was evaluated using flow cytometry by means of displacement of estradiol conjugated to fluorescein‐bovine serum albumin (E2‐BSA‐FITC) with increasing concentrations of (−)‐EPI and compared to the potent and selective GPER agonist, G1. To evaluate mitochondrial biogenesis we used fluorescence microscopy in order to quantify mitochondria density accompanied by measurement of oxidative phosphorylation complex proteins II and IV densities by using an in‐cell ELISA kit. Results indicate that (−)‐EPI is capable of displacing E2‐BSA‐FITC similarly to G1. In addition, 48 h of (−)‐EPI treatment (10 M) significantly increases mitochondrial biogenesis in C2C12 myotubes with similar efficacy to that observed with G1, effects that were partially and completely blocked by G36 (GPER antagonist), respectively. Altogether, these results indicate that GPER is expressed in muscle cells and appears to mediate to a significant extent the stimulatory effects of (−)‐EPI on mitochondrial biogenesis. Thus, GPER activation may account for the stimulatory effects on skeletal muscle structure/function observed by either cocoa or pure (−)‐EPI treatment in animals and humans.Support or Funding InformationNIH DK98717, CONACyT‐CICESE
Metoprolol prevents post‐ischemic myocardial decompensation via β3AR‐dependent protective Sphingosine‐1 phosphate signaling
Authors: Cannavo Alessandro , Rengo Giuseppe , Liccardo Daniela , Gao Ehre , Gambino Giuseppina , Pun Andres , Ibanez Borja , Leosco Dario , Ferrara Nicola , Paolocci Nazareno , Koch Walter J. 
Published: November 2023

Backgroundβ‐blockers increase survival in heart failure (HF) patients; however, the mechanisms behind this protection are not fully understood. Further, a high percentage of HF patients cannot tolerate or are non‐responders to b‐blockade. It is known that sympathetic catecholamine overactivation of β1‐adrenergic receptors (ARs) leads to sphingosine‐1‐phosphate (S1P) receptor‐1 (S1PR1) down‐regulation, contributing to HF progression since S1P‐SIPR1 signaling is cardioprotective. Hence, it is plausible that, in addition to direct β1AR blockade actions, β1AR blockers may improve decompensated heart function by restoring S1PR1 signals. Here, we tested this novel hypothesis, and sought to determine the mechanisms underlying the positive impact of β1AR blockade and S1PR1 in the ischemic heart after myocardial infarction (MI) and during HF progression.MethodsIn vitro we tested the effects of Metoprolol (Meto) in HEK293 cells and in ventricular cardiomyocytes isolated from neonatal rats (NRVMs). In vivo we assessed the effects and the potential mechanism of Meto in Wild‐type and β3AR knockout (KO) mice.ResultsHere we report that the β1AR‐blocker Meto prevents Isoproterenol‐ (βAR agonist) but not S1P‐induced S1PR1 down‐regulation in vitro. In vivo, infarcted mice treated with Meto or S1P (1 week after MI for 3 weeks) displayed attenuated cardiac function and reversed adverse LV remodeling. Combining S1P and Meto did not further improve post‐MI outcome. Previous evidence shows that Meto increases cardiac β3AR levels and activity, and in adipocytes β3AR participates in S1P secretion. Hence, we next measured cardiac Sphingosine kinase 1 (SphK1), the enzyme responsible for S1P secretion, along with circulating S1P levels, in both WT and β3AR KO mice. Circulating S1P and cardiac SphK1 protein levels were significantly reduced in MI vs. sham‐operated mice, and this effect was almost completely prevented by Meto. Conversely, β3AR KO mice displayed markedly reduced S1P and SphK1 levels, both under control and MI condition, and Meto failed to restore their levels in these animals. Congruent with this, Meto‐treatment did not ameliorate post‐MI dysfunction in β3AR KO mice. Importantly, human HF patients on β1AR‐blockers have elevated circulating S1P levels suggesting Meto is linked to S1P secretion and signaling.ConclusionsOur study uncovers a previously unrecognized mechanism by which β1‐blockers prevents disease progression in ischemic HF patients and this is dependent on b3ARs and S1P secretion. Importantly, it suggests that a mechanism of b‐blocker non‐responders may be due to β3AR‐dysfunction.Support or Funding InformationAmerican Heart Association‐GRA Winter 2016 Postdoctoral Fellowship‐16POST30980005
Functional Activity of Etrasimod and a Diverse Panel of Sphingosine‐1‐Phosphate Receptor (S1PR1) Modulators at S1P4 Receptors
Authors: Adams John W , Unett David J , Chen Xiaohua , Gaidarov Ibragim 
Published: November 2023

The S1P1 receptor modulator FTY720 (Fingolimod) exerts numerous anti‐inflammatory effects at least in part by producing sustained receptor internalization resulting in loss of cellular responsiveness to S1P. FTY720 and a number of next‐generation S1PR1 modulators also have varying degrees of activity at the S1PR4 and 5 receptor subtypes. Activity at S1PR5 has been suggested to be involved in the beneficial central effects of Fingolimod in multiple sclerosis. However, little is known about the nature of the interaction of these modulators with S1PR4. We sought to more thoroughly characterize the activity of a panel of structurally diverse S1PR4 modulators.FTY720 phosphate and a panel of S1P receptor modulators were evaluated for their abilities to interact with S1PR4 using a broad panel of functional assays designed to interrogate signaling pathways and receptor internalization. Compounds evaluated included Etrasimod (APD334), a novel S1PR modulator in clinical development, additional compounds that have undergone clinical investigation, and literature compounds reported to be S1PR4 selective. As observed with the S1P1 receptor, treatment of a S1PR4 expressing cell lines with FTY720 phosphate and structurally diverse S1PR1 modulators induced recruitment of b‐arrestin and drove rapid and robust receptor internalization. Label free Dynamic Mass Redistribution assays (Corning®, Epic®) readily detected S1PR4 mediated functional responses. Differential effects of S1P modulators on the kinetics of S1P4R internalization/recycling and persistence of signaling following drug removal were observed. Treatment with selected modulators affected the functional properties of S1PR4 expressing human dendritic cells. These data demonstrate that the pharmacology of S1PR modulators at S1PR4 largely mirrors that observed at S1PR1 and suggest that S1PR1 modulators with activity at S1PR4 may possess additional beneficial effects on immune modulation.Support or Funding InformationFunding: Arena Pharmaceuticals, Inc.
The hepatoprotective effects of prostacyclin mimetics are associated with their anti‐oxidative actions
Authors: Misawa Hiroki , Ohashi Wakana , Tomita Kengo , Shimada Yutaka , Hattori Yuichi 
Published: November 2023

Prostacyclin (PGI2) serves as a protective and anti‐inflammatory mediator. We examined whether two PGI2 mimetics, ONO‐1301 and beraprost, are beneficial in acute liver injury. Acute liver injury was induced by lipopolysaccharide/D‐galactosamine (LPS/GalN) in mice. PGI2 mimetics significantly declined the LPS/GalN‐induced increase in serum aminotransferase activity and hepatic overexpression of pro‐inflammatory cytokine genes. Histological examintaions showed that ONO‐1301 was much more effective than beraprost. The IP receptor antagonist CAY10441 abrogated their heptoprotective effects. We found that PGI2 mimetics significantly suppressed ROS generation in liver tissues of LPS/GalN‐challenged mice. Their inhibitory effects on ROS generation were also demonstrated in in vitro experiments using GalN‐ or TNF‐α‐stimulated HepG2 cells. We indicate that activation of IP receptors with PGI2 mimetics can rescue the damage in the liver induced by LPS/GalN by efficiently reducing oxidative stress. Our findings point to PGI2 mimetics as a potential novel therapeutic modality for the treatment of acute liver injury.Support or Funding Informationprostacyclin, LPS/GalN, ROS
Pyrrolizidine alkaloid‐induced hepatotoxicity by mitochondrial damage
Authors: LU YAO , Ma Jiang , Lian Wei , Fu Peter , Lin Ge 
Published: November 2023

Pyrrolizidine alkaloids (PAs) are naturally occurring heterocyclic compounds that can cause severe toxicities particularly in the liver. The reactive pyrroles formed by hepatic metabolic activation of toxic PAs can rapidly bind to intracellular proteins forming pyrrole‐protein adducts, which is deemed to be a major trigger initiating PA‐induced hepatotoxicity. Our previous proteomic study has illustrated that ATP5B, a critical subunit of ATP synthase, could be a potential target for PA‐induced hepatotoxicity. The present study aimed to further investigate the direct interaction between the reactive pyrroles and ATP5B, and also to examine the subsequent cascade of hepatotoxic events after such interaction. The immunoprecipitation of ATP5B coupled with Western blot analysis using anti‐pyrrole‐protein antibody demonstrated that reactive pyrroles covalently bind with ATP5B to form pyrrole‐ATP5B adducts in the liver of rats treated with a representative hepatotoxic PA, retrorsine, and also in the retrorsine‐treated human HepaRG hepatocytes. Further study in HepaRG hepatocytes, the ATP synthase activity were significantly reduced after the treatment of retrorsine for 6 hr. Retrorsine induced mitochondrial damage demonstrated by depolarization of mitochondrial membrane potential and dose‐dependent inhibition of mitochondrial aerobic respiration. Subsequently, at 18 hr after retrorsine treatment, cell apoptosis was evidenced by a significant elevation of caspase 3/7 activity and significantly increased numbers of apoptotic cells assessed by Annexin V/propidium iodide apoptosis assay. To the best of our knowledge, our results revealed for the first time that hepatotoxicity of PA was triggered, at least in part, by the direct interaction of ATP5B with the reactive metabolite of PA followed by impairing mitochondrial function.[Supported by RGC HKSAR (GRF Project No. 469712 and 14111816), CUHK Direct Grant (No. 4054215 and 4054302) and CUHK One‐off Funding for Joint Lab/Research Collaboration (Project Code: 3132968)].
Pulmonary Vascular Niche Regulates Macrophage Functional Polarization through Endothelial Jag1
Authors: Xing Junjie , Dai Zhiyu , Huang Xiaojia , Zhao Youyang 
Published: November 2023

BACKGROUNDThe pathogenesis of acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) have been well studied. Little is known about the mechanism and treatment for resolution of inflammatory lung injury. Especially the interaction of macrophage and pulmonary endothelium during the course of lung injury and its effect on lung injury resolution.METHODS AND RESULTSUsing jagged1 endothelial specific mice, we found that endothelial jagged1 deficiency have faster lung injury recovery than wild type mice in LPS induced lung injury. Adoptive transfer M1 macrophage impaired the repair of lung injury in jagged1 endothelial specific knockout mice. Further study showed that recruited macrophages is more M2‐like during the sepsis induced lung injury recovery phase in Jagged1 specific knockout mice. In vitro experiment we also found that inhibition of jagged1 in lung microvascular endothelial cell reprogramming macrophage to the M2 lineage and macrophage Jag1‐notch activation inhibited macrophage M2 transform.CONCLUSIONSLung EC Jagged1 mediated macrophage polarization via interaction with Notch signaling. Jag1 deletion in endothelial cell or inhibition of Jag1‐notch signal in macrophage may be the therapeutic target in resolving inflammatory lung injury.
The Cardenolide Glycoside Acovenoside A Impedes Proliferation of Lung Adenocarcinoma Cells in vitro and in vivo
Authors: Hafner Susanne , El Gaafary Menna , Schmiech Michael , Syrovets Tatiana , Simmet Thomas 
Published: November 2023

In light of the incidental and remarkable findings of the putative anticancer efficacy of digitalis glycosides in the treatment and relapse prevention of different human cancers, we have explored Acovenoside A (AcoA), a member of the cardenolide glycoside family that has been hardly investigated so far, with respect to its putative effects on tumor cells at the molecular level as well as its impact on tumor growth. Of note, at equimolar concentrations, AcoA inhibited the proliferation and induced cell cycle arrest of A549 lung cancer cells more efficiently than the approved and commonly used chemotherapeutic drug doxorubicin, whereas non‐tumorous peripheral blood mononuclear cells remained unaffected. The detection of increased amounts of caspase‐3‐ and annexin V‐positive cells indicated canonical apoptosis as the main form of the AcoA‐driven cancer cell‐specific toxicity. These in vitro findings were confirmed in a preclinical model of lung cancer xenografts grown on chorioallantoic membranes (CAMs) of fertilized chick eggs used as an in vivo model. Thus, after repeated topical application, AcoA treated xenografts expressed less green fluorescent protein and contained a significantly lower fraction of Ki‐67‐positive proliferative cells, but a higher proportion of apoptotic TdT‐positive cells indicating inhibition of proliferation and induction of apoptosis in vivo. Tumor cells loaded with a fluorescent sodium indicator exhibited an increased concentrations of cytosolic sodium ion upon AcoA treatment pointing to the inhibition of the Na+‐K+‐ATPase complex as the major cellular target of AcoA. Inhibition of the Na+‐K+‐ATPase complex in tumor cells by AcoA resulted in a reduced phosphorylation of Src kinase and Aurora kinase A playing important roles in tumor growth and metabolism. Similarly, AcoA strongly inhibited the TNF‐α induced activation of NF‐κB and the IFN‐α induced activation of the interferon regulatory factor 3 known to be involved in tumor survival pathways.Our findings on the inhibition of pathways, which are important in cancer cells, corroborate AcoA as a potent member of the cardenolide glycoside family. Based on its pharmacodynamics effects, the compound deserves further exploration as a potential anticancer drug.
Study of the Mechanisms of Jadomycin Cytotoxicity in Multidrug Resistant Triple Negative Breast Cancer Cells
Authors: Goralski Kerry B , Hall Steven R , Toulany Jay , MacLeod Jeanna , Jakeman David L 
Published: November 2023

BackgroundWe previously demonstrated that jadomycins, natural products biosynthesized by the soil bacteria Streptomyces venezuelae, are cytotoxic to drug‐sensitive and multidrug‐resistant breast cancer cells in vitro. Jadomycin cytotoxicity involves the generation of reactive oxygen species (ROS) but the downstream mechanisms contributing to cell death are not completely understood.ObjectiveTo determine if jadomycin anticancer mechanisms involve ROS‐mediated DNA damage and apoptosis in triple negative breast cancer cells in vitro.MethodsMDA‐MB‐231 control (231‐CON) and paclitaxel‐resistant (231‐TXL) breast cancer cells were treated with various concentrations of jadomycin B, F and S or the positive control drug mitoxantrone in the presence and absence of the ROS inducer auranofin or ROS inhibitor N‐acetylcysteine (NAC) for 24 to 36 hours. Western blotting for phosphorylated histone H2AX, a marker of DNA double strand breaks, was used to assess DNA damage. Annexin‐V‐FLUOS and propidium iodide flow cytometric analysis was used to measure apoptosis. The effect of jadomycin treatments on gene expression of topoisomerases IIα and IIβ (Topo IIα and IIβ, respectively) was measured using quantitative PCR. To study the direct inhibition of Topo IIα, Topo IIα decatenation of catenated circular kDNA was measured in the presence and absence of jadomycins.ResultsAll jadomycin treatments and mitoxantrone significantly increased histone H2AX phosphorylation (2.3 – 5.3‐fold) and early apoptosis (1.6 – 3.0‐fold) in 231‐CON and ‐TXL cells versus the vehicle control. NAC and auranofin did not alter jadomycin‐induced DNA damage or early apoptosis. However, auranofin did increase jadomycin‐induced necrosis/late apoptosis (2.7 – 4.5‐fold). Jadomycins caused a significant reduction of Topo IIα and IIβ mRNA expression (4.2 – 8.1‐fold decrease) and directly inhibited Topo IIα in the kDNA decatenation assays.ConclusionsJadomycins induce DNA double strand breaks and apoptosis in drug‐sensitive and multidrug‐resistant MDA‐MB‐231 breast cancer cells. These effects occur independently of ROS generation and may involve gene regulatory and functional inhibition of Topo IIα and IIβ.Support or Funding InformationThe study was funded by the Canadian Breast Cancer Foundation ‐ Atlantic Chapter, the Beatrice Hunter Cancer Research Institute, The Nova Scotia Health Research Foundation and the Dalhousie Pharmacy Endowment. S Hall was a recipient of an Izaak Walton Killam Predoctoral Scholarship. J MacLeod was a recipient of a Beatrice Hunter Cancer Research Institute Cancer Research Training Program Scholarship.