bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced oral bioavailability of quercetin by nanostructured lipid carriers (1044.24)
Authors: Sun Ming , Wang Shu , Nie Shufang , Zhang Jia 
Published: June 2021

Background: Quercetin, a flavonoid found in fruits, vegetables and grains, has been shown to have many biological activities and pharmacological actions, however, because of its high hydrophobicity, low stability, and low oral bioavailability, the clinical application of quercetin is greatly restricted. We have successful synthesized biocompatible and biodegradable quercetin loaded nanostructured lipid carriers (QNLCs) and chitosan‐coated QNLCs (CS‐QNLCs), which may improve the solubility, stability and bioavailability of quercetin.Methods: The solubility of quercetin in water and its stabilities in plasma and different gastric‐intestinal solutions were measured using a HPLC system in vitro. Native quercetin, QNLCs and CS‐QNLCs were given through intravenous or oral administration to male SD rats. Quercetin concentrations in blood at different time points were measured using a HPLC system.Results: As compared to native quercetin, QNLCs and CS‐QNLCs increased quercetin stability in plasma and gastric‐intestinal solutions. Q‐NLCs and CS‐QNLCs significantly increased AUC (area under plasma concentration‐time curve) values with prolonged blood circulation time compared with native Q.Conclusion: Nanoencapsulation significantly increased quercetin aqueous solubility, stabilities in plasma and gastric‐intestinal solutions, and bioavailability.Grant Funding Source: Texas Tech University
A grape seed procyanidin extract lowers serum triglyceride levels via selective modulation of intestinal FXR‐target gene expression and inhibition of enterohepatic bile acid recirculation (1045.35)
Authors: Hintz Rebecca , Caiozzi Gianella , Ricketts Marie‐Louise 
Published: June 2021

As ligands of the farnesoid x receptor (FXR), bile acids (BA) play an essential role in maintaining triglyceride (TG) homeostasis. We previously showed that a grape seed procyanidin extract (GSPE) decreases serum TG levels via repression of sterol regulatory element binding protein 1c (SREBP1c) in a FXR‐dependent manner. We now propose an additional novel mechanism by which GSPE lowers TG levels, wherein GSPE selectively modulates intestinal FXR‐target gene expression, therefore altering enterohepatic BA recirculation. Studies were performed using human colorectal adenocarcinoma cells (Caco2) and wild type (WT) and FXR‐/‐ mice. Results, from both in vitro and in vivo studies, show that GSPE represses the expression of the apical sodium‐dependent bile acid transporter (ASBT), while also inhibiting the expression of intestinal bile acid‐binding protein (IBABP), thereby impacting BA uptake and transport through the enterocyte. Concomitantly, a 43% decrease in serum BA levels was observed in vivo following GSPE administration in WT mice. Hepatic CYP7A1, the gene encoding cholesterol 7α‐hydroxylase, the rate‐limiting enzyme in the classical BA biosynthetic pathway was increased; while the expression of ileal fibroblast growth factor 15 (FGF‐15), the critical gut‐liver regulator of CYP7A1 and BA synthesis was repressed. In addition, serum TG levels were significantly decreased by 30% post‐GSPE administration. Based on our results, we propose that GSPE acts as an intestinal gene selective bile acid receptor modulator (BARM), thereby altering enterohepatic BA recirculation, which contributes to the TG‐lowering ability of this extract. Grant Funding Source: Supported by USDA‐Hatch NEV0738 and Multi‐state project W3122
The antiatherogenic effects of targeted epigallocatechin gallate ‐ loaded nanoparticles (1045.41)
Authors: Zhang Jia , Nie shufang , Wang shu 
Published: June 2021

Objective: The objectives of this study were to incorporate scavenger receptor target ligands on the surface of biocompatible and biodegradable epigallocatechin gallate (EGCG)‐loaded nanoparticles (E‐LNP), to evaluate their target specificity to macrophages in vitro and atherosclerotic lesions in LDLr‐/‐ mice.Method: E‐LNP were synthesized using a sonication method. Particle size, zeta potential and morphology were determined using a transmission electron microscope and Brookhaven analyzer. The target specificity to macrophages (in vitro) and to atherosclerotic lesions in LDLr‐/‐ mice (in vivo) was measured using a fluorescence microscopy and an IVIS® Lumina XR imaging system, respectively. Macrophage cholesterol content and EGCG uptake were measured using a HPLC method. After intravenous injection of E‐LNP once per week for 20 weeks, the aortic lesion was quantified by a microscope and image J software.Results— E‐LNP are negatively charged and less than 100 nm in diameter. As compared to nanoparticles without target ligands, targeted E‐LNP significantly increased macrophage EGCG content and decreased macrophage expression of monocyte chemoattractant protein 1 (MCP‐1). The target ligands significantly increased the binding and uptake of those nanoparticles to THP‐1 derived macrophages and improved the target specificity to atherosclerotic lesions in LDLr‐/‐ mice.Conclusions— Targeted E‐LNP have a potential for decreasing aortic cholesterol accumulation and inflammatory responses through targeting to macrophages and foam cells in atherosclerotic lesions.Grant Funding Source: Supported by NIH 1R15AT007013‐01
Protease‐activated peptide toxins for selective nanoparticle therapeutics (1054.4)
Authors: Jallouk Andrew , Palekar Rohun , Schlesinger Paul , Pan Hua , Wickline Samuel 
Published: June 2021

Objective: To enhance peptide toxin specificity by developing prodrugs which are locally activated by proteases and can be delivered by antigen‐targeted nanoparticles.Introduction: Melittin is a peptide toxin derived from bee venom which has been suggested as a cancer therapy due to its membranolytic activity. Yet, its therapeutic applications have been limited by its rapid clearance from the circulation and nonspecific cytotoxicity. We have previously developed an integrin‐targeted nanoparticle system which extends melittin circulation time and enhances delivery to tumors. Here, we present a platform for melittin prodrug design which allows activation by a protease of interest while still permitting delivery of the prodrug by nanoparticle carriers.Methods and Results: The prodrug consisted of melittin joined to a blocking sequence via a matrix metalloproteinase 2 (MMP‐2)‐cleavable linker. While native melittin was substantially hemolytic (HD50: 5.4 µM) and cytotoxic (IC50: 3.0 µM) to B16 melanoma cells, the prodrug demonstrated no hemolysis (HD50: > 100 µM) and only slight cytotoxicity (IC50: > 100µM) at concentrations up to 100 µM. MMP‐2 cleavage restored the hemolytic activity (HD50: 11.2 µM) and cytotoxicity (IC50: 4.8 µM) of the prodrug. Surface plasmon resonance spectroscopy confirmed the ability of the prodrug to insert into the lipid monolayer of perfluorocarbon nanoparticles.Conclusion: Protease‐activated melittin prodrugs minimize nonspecific cytotoxicity and show promise for development as selective nanoparticle‐based therapeutics.Grant Funding Source: Supported by a grant from the National Institutes of Health (R01 HL073646 to SAW)
Mode and site of interaction of the novel antiplatelet drug ticagrelor at the recombinant human P2Y12 receptor (1057.11)
Authors: von Kügelgen Ivar , Lutz Dominique , Straßburger Jens , Baqi Younis , Müller Christa , Hoffmann Kristina 
Published: June 2021

Ticagrelor is a perorally available P2Y12 receptor antagonist used for the prevention of cardiovascular events. In the present study, we analyzed the mode of action of ticagrelor at the recombinant human P2Y12 receptor and mutant constructs stably expressed in CHO Flp‐In cells. ADP and its analogue 2‐methylthio‐ADP (2‐MeSADP) decreased forskolin‐induced cAMP‐accumulation in a concentration dependent manner with 2‐MeSADP being more potent than ADP. Ticagrelor (3 to 10 nM) itself did not change basal and forskolin‐induced cAMP‐levels. Addition of ticagrelor at increasing concentrations led to increasing rightward shifts of the concentration‐response curves of ADP and 2‐MeSADP. An analysis according to Schild revealed pA2‐values of 8.7 with slopes of the linear regression lines not different from unity. These data are in agreement with a competitive mode of antagonism of ticagrelor against both agonists. The antagonistic potency of ticagrelor was maintained in cells expressing S101A‐, K173A/K174A‐, C248A‐, R256A‐ and K280A‐mutant constructs. In contrast, there were no rightward shifts of the concentration‐response curves of 2‐MeSADP and ADP by ticagrelor in cells expressing the C194A‐mutant P2Y12 receptor construct. Interestingly, antagonistic potencies of reactive blue 2 and analogues including PSB‐0739 (1‐amino‐4‐[4‐phenylamino‐3‐sulfophenylamino]‐9,10‐dioxo‐9,10‐dihydroanthracene‐2‐sulfonate) were increased in cells expressing the C194A‐mutant construct. In summary, our results suggest a competitive mode of antagonism of ticagrelor at the human P2Y12 receptor. The residue Cys194 (located in transmembrane region 5) appears to belong to the binding site of ticagrelor within the P2Y12 receptor protein.Grant Funding Source: Supported by the University of Bonn
Development and anti‐tumor activities of claudin‐4‐specific monoclonal antibodies (1062.8)
Authors: Kawahigashi Yumi , Li Xiangru , Kuniyasu Hiroki , Fukasawa Masayoshi , Tada Minoru , Ishii Akiko , Watari Akihiro , Yagi Kiyohito , Kondoh Masuo 
Published: June 2021

Claudin (CLDN) is a key component of the epithelial tight junction. Expression of the various CLDNs differs among tissues and is deregulated in malignant tumors. For example, CLDN4 is frequently overexpressed in pancreatic, breast, and ovarian cancers and therefore may be an attractive target for anticancer drugs. However, the low antigenicity of CLDN4 has hampered the development of antibody‐based CLDN4‐targeted cancer therapy. We successfully generated monoclonal antibodies (mAbs) to CLDN4 and evaluated their anti‐tumor activity. Briefly, we immunized rats with plasmids containing the CLDN4 coding sequence. We fused the resulting B cells with myeloma cells and screened 8 hybridoma clones that produced anti‐CLDN4 mAbs. All 8 mAbs bound to CLDN4‐expressing cells but not to those expressing CLDN1, 2, 5, 6, 7or 9. All 8 mAbs showed CLDN4 binding‐dependent activation of the Fcγ receptor. A clone with high affinity to CLDN4 suppressed tumor growth in vitro and in vivo. Interestingly, treatment of cancer cells with the clone of CLDN4‐specific mAb and an anti‐cancer drug (5‐flurouracil or cisplatin) had a synergistic cytotoxic effect. Specifically, mAb treatment increased intracellular drug levels. Our findings indicate that CLDN4 mAbs are promising tools for CLDN4‐targeted cancer therapy.Grant Funding Source: supported by the Ministry of Health, Labor, and Welfare of Japan
Investigation into the predominate mechanism of OAT1 inhibition (1064.15)
Authors: Pelis Ryan , Ingraham Leslie , Li Mansong 
Published: June 2021

Renal tubular drug secretion mediated by the organic anion transporter 1 (OAT1) is a potential site of drug‐drug interactions. A number of marketed drugs inhibit OAT1, but the mechanism by which they do so is not typically known. The purpose was to investigate the predominant mechanism by which therapeutic drugs inhibit OAT1. OAT1 was expressed in Chinese hamster ovary cells and 3H‐para‐aminohippurate was used as the substrate. The Kreb’s cycle intermediate alpha‐ketoglutarate (αKG) competitively inhibited OAT1 with a Ki value (5.4 μM) approximating its unbound plasma concentration. In the presence of αKG (5 μM) the potency of inhibition caused by probenecid, furosemide and ibuprofen decreased ~2‐fold (~2‐fold increase in IC50 values), whereas the potency of inhibition caused by telmisartan was unaffected. This led us to speculate that probenecid, furosemide and ibuprofen interact competitively with αKG and 3H‐para‐aminohippurate while telmisartan is a non‐competitive inhibitor. Probenecid, furosemide and ibuprofen all competitively inhibited OAT1 with Ki values of 10.5 μM, 17.8 μM and 3.1 μM, respectively. In contrast, telmisartan inhibited OAT1 by reducing the maximal transport rate without affecting the Michaelis constant, indicating a non‐competitive type inhibition (IC50 of value of 0.33 μM). Interestingly, wash‐out experiments showed that the inhibitory effect of telmisartan is quasi‐irreversible. These data show that OAT1 inhibition can occur through different kinetic mechanisms. The mechanism by which inhibitors reduce OAT1 activity could influence the magnitude of drug‐drug interactions at OAT1‐mediated renal tubular drug secretion. Supported by NSHRF.Grant Funding Source: Supported by Nova Scotia Health Research Foundation
Sequential hydrolysis of dabigatran etexilate to its active metabolite by human carboxylesterase 1 and 2 (1064.6)
Authors: Hu Zheyi , Parker Robert , Herring Vanessa , Laizure S. 
Published: June 2021

The direct thrombin inhibitor dabigatran etexilate (DABE) is a double prodrug that undergoes near complete presystemic conversion to the active dabigatran (DAB) metabolite after oral dosing in humans. Based on the ethyl ester and carbamate ester groups in its structure and previous reports that DABE is hydrolyzed by serine esterases, we hypothesized that human carboxylesterase‐1 (CES1) and carboxylesterase 2 (CES2) are responsible for the hydrolysis of DABE to DAB. DABE enzyme kinetics in human recombinant CES1 and CES2, human intestinal microsomes (HIM) and human liver S9 fractions (HLS9) were performed. Sequential hydrolysis of DABE in HIM and HLS9 was also conducted to simulate the in vivo disposition of DABE after oral administration. We found that hydrolysis of the DABE double prodrug by both CES1 (ethyl ester) and CES2 (carbamate ester) is required for the formation of DAB and that complete conversion of DABE to DAB was achieved by sequential hydrolysis in HIM and then HLS9. Based on this scheme, we propose that the major pathway responsible for the presystemic formation of DAB is hydrolysis of the DABE carbamate ester by CES2 in the intestines followed by conversion to DAB in the liver by CES1‐mediated ester hydrolysis. This proposed pathway is consistent with the near total presystemic conversion of DABE to DAB and the sequential exposure to CES2 and CES1 following oral dosing based on high expression levels of CES2 and CES1 in the intestines and liver, respectively.Grant Funding Source: National Institutes of Health National Institute of General Medical Sciences
Target of selective cyclic nucleotide phosphodiesterases differentially regulates arterial myocyte migration (1065.3)
Authors: Brzezinska Paulina , Hubert Fabien , Rampersad Sarah , Wudwud Alie , Freitag Silja , Umana M , Maurice Donald 
Published: June 2021

Vascular myocyte (VSMC) migration is a vital process allowing for the regulation of vascular development, tissue repair and the modulation of pathophysiological conditions including atherosclerosis. Atherosclerosis induced damage to the vascular endothelium results in medial VSMC migration into the intima to promote vessel repair. If unregulated, it may exacerbate atherosclerosis related stenosis. cAMP is a regulator of VSMC migration and cAMP‐elevating agents inhibit VSMC migration in vitro and reduce neointimal thickening in vivo. However, these agents promote the formation of integrin‐based adhesions and leading edge structures, which are necessary events for directional cellular migration. The localization of several distinct pools of cAMP generated by distinct cyclic nucleotide phosphodiesterases (PDEs) may explain the differential effects of VSMC migration regulated by cAMP. We used a modified migration assay to visualize and quantitate the formation of leading edge structures by VSMCs during migration. Here, we report differential migration related effects through the target of selective cAMP pools generated by distinct PDEs.Grant Funding Source: Canadian Institutes of Health Research
Concentration‐dependent effects of zinc on angiotensin‐converting enzyme‐2 activity (1067.4)
Authors: Speth Robert , Carrera Eduardo , Jean‐Baptiste Malaika , Joachim Arline , Linares Andrea 
Published: June 2021

Angiotensin‐converting enzyme‐2 (ACE‐2) degrades angiotensin II (Ang II) and forms Ang (1‐7), which antagonizes much of the pathophysiology of Ang II. ACE‐2, a member of the M2 family of metallopeptidases, contains a HEXXH motif that functions as the zinc binding domain at its active site. To ascertain the importance of zinc on ACE‐2 activity, we measured metabolism of an artificial substrate of ACE‐2 (MCA‐APK‐Dnp) by rat kidney and lung, as well as recombinant human ACE‐2 (rhACE‐2) at various concentrations of zinc. Frozen rat tissues were homogenized in 19 volumes of 20 mM NaPO4 at pH 7.15 with 0.05% Triton X‐100 detergent. The 48,000 x g supernatant was diluted 40‐fold in assay buffer, final concentrations: 50 mM sodium phosphate (pH 7.0); 100 mM NaCl; 0, 10, 100, or 1000 µM zinc acetate; with or without the ACE‐2 inhibitor MLN‐4760. rhACE‐2 was present at a concentration of 4 pg/µL. Metabolism of the fluorogenic substrate (50 µM) was measured at 393 nm with excitation at 328 nm at 37oC. In both rat tissues and rhACE‐2 the rate of MLN‐4760‐blockable metabolism of substrate was highest with no zinc or 10 µM zinc. However, in the presence of 100 µM zinc, activity was significantly (p<0.05) decreased in rat lung and rhACE‐2 compared to 0 or 10 µM zinc. In the presence of 1000 µM zinc, activity was further reduced (p<0.05) in all three preparations compared to 0, 10 and 100 µM zinc. These results suggest that ACE‐2 may have additional lower affinity binding site(s) for zinc that interfere with its ability to metabolize its substrates. Assays of ACE‐2 activity should not be run in the presence of zinc concentrations greater than 10 µM.Grant Funding Source: NIH‐HL113905
Enhancement of dynein‐mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes (1077.10)
Authors: Xu Ming , Li Xiaoxue , Chen Yang , Zhang Yang , Li Pin‐lan 
Published: June 2021

We have recently demonstrated that dynein‐mediated autophagosome (AP) trafficking contributes to the formation of autophagolysosomes (APLs) and autophagic flux process in coronary arterial myocytes (CAMs). It remains unknown how the function of dynein as a motor protein for AP trafficking is regulated under physiological and pathological conditions. The present study tested a hypothesis that the dynein‐mediated autophagy maturation is regulated by a redox signaling mechanism associated with NADPH oxidase. First, we showed that in primary cultures of CAMs reactive oxygen species (ROS) including H2O2 and O2‐. generated by xanthine/xanthine oxidase (X/XO) significantly increased APs as shown by largely increased level of LC3B, an AP marker. It was also found that ROS dramatically increased the number of APLs in CAMs as shown by flow cytometic analysis of APLs using acridine orange. Furthermore, fluorescent confocal microscopy demonstrated that both ROS increased the fusion of APs with lysosomes in CAMs as detected by increased colocalization of LC3B with lysosome marker Lamp1. All these changes in APs formation and autophagic flux were also enhanced by overexpression of NADPH oxidase‐1 (Nox1) gene in CAMs after cDNA transfection. Mechanistically, we found that enhanced autophagic flux by ROS was associated with activation of the motor protein‐dynein, which increased APs movement in CAMs. This ROS‐induced APs movement was abolished by either inhibition of dynein activity with EHNA or disruption of the dynein complex. Lastly, another stimulus, high glucose, was used to increase the APLs formation in CAMs, which was abolished by either Nox1 inhibitor, ML117 or dynein inhibitor, EHNA. Taken together, these data suggest that ROS increases autophagic flux through enhanced dynein‐dependent APs trafficking in CAMs.Grant Funding Source: HL057244, HL091464 and HL075316
Natriuretic responses to angiotensin 1‐7 infusion in hypertensive rat models (1088.6)
Authors: Barry Elaine , Johns Edward 
Published: June 2021

Anti‐natriuretic properties of Angiotensin II in the kidney are counterbalanced by the opposing actions of Angiotensin 1‐7 (Ang 1‐7) in normal animals. This study investigated whether the response to Ang 1‐7 was altered in models of hypertension where the renin‐angiotensin system (RAS) was either suppressed (Deoxycorticosterone (DOCA) salt hypertensive) or enhanced (2‐Kidney‐1‐clip (2K1C) hypertensive). Hypertensive groups derived from male Wistar rats (n=4) were anaesthetised and prepared for renal interstitial infusion of Ang 1‐7 (3x10‐³ M) at 1ml/h into the left kidney and cannulation of its ureter. Data±SEM were subject to one‐way ANOVA. Ang 1‐7 had no effect on blood pressure, at 139mmHg for 2K1C rats and 154mmHg for DOCA Hypertensive rats. In 2K1C rats, sodium excretion (UVNa) increased from 6.1±1.4 to 8.7±2.2µmol/min and fractional sodium excretion (FENa) from 1.2±0.2 to 1.5±0.2%, similar to normal rats. Intra‐renal infusion of the same dose of Ang 1‐7 in the DOCA model has no effect on the excretory variables showing a statistically significant difference in the response of these two models. These data suggest that the action of Ang(1‐7) correlates with the level of RAS activation.Grant Funding Source: Supported by Science Foundation Ireland (SFI)
Model of chloride‐bicarbonate counter‐movement in CO2‐sensitive avian intrapulmonary chemoreceptors (1092.2)
Authors: Lonjaret Jean‐Guillaume , Bassett Peter , Meehan Shane , Sheridan Krystal , Hempleman Steven 
Published: June 2021

Our working model of IPC CO2 transduction (AJP, 279: R1988‐1995, 2000) posited intracellular H+ as the transduced variable, with H+ critically dependent on carbonic anhydrase, rapid H+ buffering, and transmembrane H+ exchange. Empirical testing has refined the model (and vice versa), and recent experiments suggest that Cl‐ ions, currently ignored in model, have a significant role in IPC CO2 transduction. In‐vivo Cl‐ ion channel blockers (eg. niflumic acid), and Cl‐ transport inhibitors (bumetanide, furosemide, DIDS) all significantly affect IPC response to CO2. We therefore modified the IPC model to include rapid, bidirectional HCO3‐/Cl‐ channel and transporter fluxes, and cation‐coupled Cl‐ transport, similar to the HCO3‐/Cl‐ shift in RBCs. Net Cl‐ and HCO3‐ transmembrane fluxes during CO2 stimulation were constrained by macroscopic electroneutrality. The new model predicts net Cl‐ influx and HCO3‐ efflux across IPC membrane when PCO2 is high, and net Cl‐ efflux and HCO3‐ influx when PCO2 is low; both affect IPC excitability. A system linked differential equations was formulated, then solved using Berkeley Madonna 8.3.18 software. Results indicate that large, phasic transmembrane HCO3‐/Cl‐ fluxes are required for normal IPC CO2 transduction.Grant Funding Source: supported by NIH 2R15 HL087269‐02