bluuebunny/crossref_metadata_2025 huggingface dataset:
Vegetable consumption and associated nutrient intakes in the United States: results from NHANES 2009‐10 and the new USDA Food Patterns Equivalents Database (1023.5)
Authors: Gugger Carolyn , Bidwai Samruddhi , Joshi Nandan , Holschuh Norton , Albertson Ann
Published: June 2021

This study examined associations between vegetable consumption, and nutrient intake among the American population. Using 1‐day dietary recall data from NHANES 2009‐10 and the new USDA Food Patterns Equivalent Database (FPED), mean daily vegetable (VEG) intake, percent not meeting recommendations and major food sources were identified for children aged 2‐18 yr (n=3124) and adults 19+ yr (n=5820). Further, these populations were classified into groups based on their VEG consumption for Adults: <=1 (43%), >1‐2 (32%), or >2 (25%) servings and for Kids: <=0.5 (41.5%), >0.5‐1 (27.5%), or >1 (31%). On average, children and adults consumed 0.89 and 1.59 cup equivalents of VEG/d, respectively. Only 3% and 13.3% percent of children and adults, respectively, met the MyPlate recommendations for vegetable intake. French fried potatoes were the leading source of VEG at 15.4% for children, followed by lettuce (12.6%) and tomatoes (8.7%). For adults, lettuce (14.5%), tomatoes (13.9%) and onions (6.8%) were top sources. Children consuming greater than >1 VEG, and adults consuming >2 VEG had significantly higher nutrient intakes, except for vitamin D. In conclusion, consumption of vegetables by Americans remains well below the recommended level. Approaches to increase vegetable consumption within current energy needs should continue to be emphasized, particularly for children.
The effectivness of daily consumption of 50 g dried plum on improving indices of bone turnover in osteopenic postmenopausal women (1027.5)
Authors: Metti Dina , Ortiz Dawn , Cravinho Amanda , Vereda Yenina , Garcia Sofia , Clayton Zach , Kern Mark , Arjmandi Bahram , Hooshmand Shirin
Published: June 2021

Our findings in osteopenic postmenopausal women indicated that daily consumption of 100 g dried plum for one year is highly effective in increasing bone mineral density as well as improving the indices of bone turnover. The objective of our current study was to examine whether 50 g dried plum will be as effective as 100 g dried plum in reversing bone loss in osteopenic older postmenopausal women. Forty five osteopenic 65 to 79 year old women were randomly assigned into one of the three groups: 1) 50 g dried plum; 2) 100 g dried plum; and 3) control. All groups received 500 mg calcium and 400 IU vitamin D daily. Blood samples were collected at baseline, 3‐, and 6‐month to assess bone biomarkers and bone metabolism measurements. Physical activity recall and 3‐day food records were obtained at baseline, 3‐, and 6‐month to examine physical activity and dietary confounders as potential covariates. Initial preliminary analyses of bone specific alkaline phosphatase (BAP; a marker of bone formation) showed increase at 3‐months and 6‐months in 50 g and 100 g dried plum groups. Interestingly, tartrate resistant acid phosphatase‐5b (TRAP5b, a marker of bone resorption) decreased at 3‐months and 6‐months intervals in 50 g and 100 g dried plum groups. These results even though preliminary suggest that 50 g dried plum possibly could be as effective as 100 g dried plum in reversing bone loss in older osteopenic postmenopausal women.
Heat stress during differentiation of porcine adipocytes is accompanied by increased expression of adipocyte marker genes and downregulation of inflammatory genes (1033.2)
Authors: Lu Hang , Wang Henxiao , Ajuwon Kolapo
Published: June 2021

Global increase in ambient temperature as a result of climate change imposes heat stress on both humans and livestock. For pigs, an important food animal and a biomedical model, the implication of sustained heat stress on growth, physiology and metabolism of the animal is still unclear. Because of the role that adipose tissue plays in the regulation of overall metabolism and growth efficiency in the pig, this study was conducted with the objective of determining the impact of heat stress during adipocyte differentiation on expression of mature adipocyte and inflammatory marker genes. Preadipocytes isolated from the subcutaneous adipose depot were differentiated under normal (37oC) or heat stress (41.5oC) condition for 3, 6 or 9 days. Gene expression was determined by RT‐PCR. Fold expression of genes at 41.5oC was determined relative to 37oC after normalization with 18s. Heat stress induced 1.5 and 1.3 fold increase in the expression of PPARγ on days 3 and 6, with no change on day 9. Expression of CEBPα was induced by heat stress by 2.4, 2.6 and 2.2 fold on days 3, 6 and 9 respectively. Expression of adiponectin (3.5, 1.9 and 3.3 fold), leptin (2.0, 2.7 and 2.3 fold), HSP70 (2.9, 3.5 and 2.2 fold), HSP90 (2.7, 5.2 and 4.5 fold) was higher in heat stressed adipocytes on days 3, 6 and 9 respectively. However, heat stress led to a reduction in the expression of IL‐6 (0.3, 0.3 and 0.2 fold) and TNFα (0.3, 0.5 and 0.1 fold) on days 3, 6 and 9 respectively, relative to cells differentiated at 37 oC. Therefore, heat stress leads to significant alteration in adipocyte metabolism in a way that may be consequential for the health and growth performance of pigs.
Elucidating a mechanism for maltooligosacchride sensing at the small intestine enterocyte (1039.9)
Authors: Chegeni Mohammad , Kim Choon Young , Naim Hassan , Hamaker Bruce
Published: June 2021

As the primary products of starch digestion by pancreatic α‐amylase, maltose and maltooligosaccharides, are the dominant substrates for α‐glucosidases at the intestinal brush border. Here, immunobloting and biosynthetic labeling using [35S]‐methionine revealed maltose induces the formation of a higher molecular weight (HMW) sucrose‐isomaltase (SI) species with a higher rate for early trafficking and maturation which is more associated with Triton X‐100 lipid rafts, resulting in higher apical sorting of these species. As this finding is potentially indicative of a maltose sensing ability by enterocytes, our goal is to investigate the sensing mechanism on the small intestine enterocytes for maltose sensing leading to the processing of HMW SI. Mass spectrometry study and endoglycosidase treatment of HMW SI showed the increased molecular weight of SI is a consequence of altered N‐ and O‐glycosylation in the Golgi apparatus causing higher apical sorting of SI. In addition, metabolomics study on metabolites involved in differentiation along with transepithelial electric resistance and endoplasmic reticulum stress measurements reveled cells maintain their differentiation through an adaptation to utilize maltose as an energy source. Thus, maltose sensing ability of enterocytes influences the intracellular processing of SI, speculatively to enhance their digestive property.
Mineral content and in vitro iron bioavailability in seaweeds (1042.7)
Authors: Dunn Michael , Flores Shireen , Dobbs Joannie
Published: June 2021

Iron (Fe) deficiency remains a public health problem despite large‐scale intervention programs. Deficiency is often due to low iron intake and/or bioavailability, especially in plant‐based diets. With the Dietary Guidelines recommending a shift toward plant‐based diets, there is increased need to identify plant foods that are good sources of iron. Seaweeds are known to be rich in iron, but its bioavailability is poorly understood. Our objective was to evaluate the quality of seaweeds as an iron source by comparing their iron content and bioavailability to spinach. Seaweeds were obtained from commercial sources. Mineral assay by ICP‐ES showed that 10 of 13 seaweeds analyzed contained more Fe/g dry matter (DM) than spinach. Iron content in the seaweeds ranged from 73 to 3490 ug/g DM compared to 166 ug/g DM in spinach. Because of the high Fe content, most seaweeds could be considered good sources of iron based on a 10 g DM serving, e.g. 100% of the Daily Value of iron could be obtained from just 5.2 g of dulse powder, 2.3 g of dried sea lettuce, or 6 g of dried nori (one sheet). However, despite their high Fe content, bioavailability studies using the in vitro digestion/Caco‐2 cell model indicated that many seaweeds do not provide more bioavailable Fe per g DM than spinach. This was due to low Fe absorption efficiencies in the seaweeds. Exceptions were nori and sea lettuce which provided 3 and 5‐fold more bioavailable Fe per g DM than spinach, respectively. The higher bioavailability resulted from a combination of higher absorption efficiencies and higher Fe contents than spinach. In addition, vitamin C increased Fe bioavailability in these two seaweeds by over 100%. Some seaweed samples were found to contain high levels of aluminum (2 mg/g DM), arsenic (66 ug/g DM), or manganese (500 ug/g DM). The level of these contaminants should be considered when recommending seaweeds for regular consumption. We conclude that certain seaweeds could be good sources of iron provided that levels of potentially toxic minerals are low.

Grant Funding Source: Supported by USDA/Hatch HAW000279‐H
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
LC‐MS/MS method development for the measurement of 15 estrogens and estrogen metabolites in human plasma and serum (1048.10)
Authors: Wambua Dickson , Craft Neal , Ziegler Regina
Published: June 2021

Circulating estradiol is strongly associated with increased risk of postmenopausal breast cancer. Estrogen metabolism profiles may also play an important role, but hypotheses could not be tested since assays appropriate for large population‐based studies were not available. Aim: To develop an accurate, fast, sensitive LC‐MS/MS method for quantification of the 15 circulating estrogens and estrogen metabolites (jointly EM). Methods: The basic method for unconjugated EM involves spiking the sample with stable isotopically labeled EM standards, organic extraction, dansyl chloride derivatization, and analysis by LC‐MS/MS HESI in the selected reaction monitoring scan mode. An enzymatic hydrolysis step permits the measurement of total (unconjugated + glucuronidated + sulfated) EM. A 100+ minute published LC separation was compared with three other LC methods using methanol or acetonitrile in the mobile phase. Also, various methods of extracting EM including liquid‐liquid, SPE, solid supported liquid (diatomaceous earth), and molecular imprinted polymer, were evaluated for efficiency and selectivity. Results: The 15 EM could be separated by LC in less than 11 minutes. The cleanliness of the extracts for several EM, as measured by signal to noise ratio, was improved >100‐fold. The resulting method is applicable to both serum and heparin‐plasma in large population studies.
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
Regulation of phenotype‐associated phosphodiesterase expression in human vascular smooth muscle cells by co‐culture with endothelial cells: role of shear stress (1065.6)
Authors: Hubert Fabien , Brzezinska Paulina , Rampersad Sarah , Maurice Donald
Published: June 2021

Vascular smooth muscle cells (VSMCs) are differentiated and quiescent (contracile phenotype) in adult blood vessels. In contrast, VSMCs can become activated and highly proliferative and migratory (synthetic phenotype) under certain pathophysiological conditions such as atherosclerosis, or when medial VSMCs are isolated and purified for cell culture. Studies have established that cAMP relaxes contractile VSMCs and inhibits the proliferation and migration of synthetic VSMCs. Furthermore, we previously showed that modification of the expression and activity of phosphodiesterases (PDE), the cyclic nucleotide hydrolysing enzymes, accompanies the phenotypic switch associated with culturing human VSMCs. Vascular endotheliall cells (VECs) can also promote a differentiated VSMC phenotype given their close proximity to VSMCs in vivo. In this context, and focusing on cAMP‐signaling pathways, we are studying the impact of VEC‐VSMC interactions on VSMC phenotypic modulation as well as the effects of shear stress. Our results indicate that VEC‐VSMC co‐culture and laminar shear stress impact phenotype‐associated PDE expression in human VSMCs.
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
Molecular mechanism of Rac1 regulation of endothelial permeability (1095.4)
Authors: Daneshjou Nazila , Komarova Yulia , Sieracki Nathan , Schwartz Martin , Malik Asrar
Published: June 2021

The RhoGTPase Rac1 plays a critical role in regulating endothelial junctional permeability; however, the causal relationship between Rac1 activity at adherens junctions (AJs) and stability of Vascular Endothelial (VE)‐cadherin adhesion, the main adhesive complex of AJs, is not well understood. Here using photo‐activatable (PA)‐Rac1 and VE‐cadherin‐Dendra2 reporter, we studied Rac1‐mediated VE‐cadherin dynamics. We observed that activation of Rac1 reduced VE‐cadherin dissociation from AJs but had no effect on VE‐cadherin association. The net result of Rac1 activation was to promote steady‐state accumulation of VE‐cadherin at AJs by increasing the affinity of VE‐cadherin homotypic interaction. Inhibition of VE‐cadherin‐mediated adhesion with a peptide that prevents formation of VE‐cadherin‐trans interaction abolished Rac1‐mediated effect on VE‐cadherin kinetics, suggesting that stabilization of trans interaction is required for Rac1 to re‐anneal the AJ barrier. Importantly, Rac1 counterbalanced RhoA activity and decreased the mechanical tension applied to VE‐cadherin adhesion as measured using FRET biosensor. Similar to Rac1 activation, inhibition of Rho kinase (ROCK) at AJs, by un‐caging ROCK inhibitor in UV‐dependent manner, led to accumulation of VE‐cadherin at AJs via stabilization of VE‐cadherin‐trans interaction. Our findings elucidate the important molecular functions of Rac1 in promoting AJ re‐annealing through stabilizing VE‐cadherin‐trans interaction.

Grant Funding Source: Supported by AHA 13PRE14670018 and NIH R01 HL045638‐24
The novel Mas agonist, CGEN 856S, promotes Akt/eNOS activation and anti‐proliferation with FOXO1 involvement (1097.3)
Authors: Peluso Antonio , Passos‐Silva Danielle , Etelvino Gisele , Rotman Galit , Santos Robson
Published: June 2021

Biotechnology advances allowed the discovery of a potential Mas agonist, CGEN 856S, which produces several effects resembling those produced by Angiotensin‐(1‐7). This study evaluated the effect of CGEN 856S on FOXO1 and Akt/eNOS activation, using different cell types. HAEC (Human Aortic Endothelial Cell), A549 and DU145 (human tumoral linages from lungs and prostate, respectively) cell were treated with CGEN 856S with or without previous treatment with A779. The cells were used for immunolocalization of FOXO1, analyzed by confocal microscopy and the nuclear fluorescence intensity was quantified. Mas‐transfected CHO (chinese hamster ovary) cells were stimulated with CGEN 856S. Untransfected CHO cells were used as control and the proteins were used for phospho‐Akt, phospho‐eNOS, phosphor‐FOXO1, Mas and GAPDH detection by western blot. A549 and DU145 cells were also used for cytotoxicity test, being treated with CGEN856S with a combination of PI3 kinase inhibitors, Wortmannin or LY294002, in different concentrations. The results showed a significant nuclear translocation of FOXO1 in HAEC, A549 and DU145 cell types treated with CGEN 856S. The Mas antagonist A779 attenuated to a great extent this effect. The phosphorylation of Akt, eNOS as well as the dephosphorylation of FOXO1 in CHO‐Mas cells treated with CGEN856S was significant. In both DU145 and A549 cells, the combination of the PI3 kinase inhibitors with CGEN 856S was able to potentiate it anti‐proliferative effect. These data suggest that CGEN 856S induces its effects similarly to Ang‐(1‐7), via Mas receptor.

Grant Funding Source: Supported by CNPq/MCTI/FAPEMIG #573924/2008‐2
Distinguishing HCO3‐ from CO3= transport by the electrogenic Na/HCO3 cotransporter NBCe1 (SLC4A4) (1098.7)
Authors: Moss Fraser , Lee Seong , Parker Mark , Boron Walter
Published: June 2021

Theoretically, the electrogenic NBCe1 (SLC4A4), could operate by five thermodynamically equivalent mechanisms: (1) 1 Na+ + 2 HCO3‐, (2) 1 Na+ + 1 CO3=, (3) NaCO3‐ ion pair uptake, (4) 1 Na+ + 1 HCO3‐ uptake with 1 H+ extruded, and (5) CO2/HCO3‐‐stimulated extrusion of 2 H+. We obtained current‐voltage (I‐V) relationships from NBCe1 expressing oocytes superfused with out‐of‐equilibrium (OOE) solutions that vary extracellular pH (pHo) from 9.0 to 6.0 at a constant [CO2]o and [HCO3‐]o. Ignoring direct pHo effects on NBCe1, #1 predicts under constant [CO2]o & [HCO3‐]o, Erev will not change. #4 & #5 predict graded decreases in pHo produce steep graded decreases in H+ efflux that manifest as steep graded decreases in slope conductance (GNBC) and inward rectification. #2 & #3 predict pHo decreases will act by lowering [CO3=]o and [NaCO3‐]o, thereby reducing GNBC as predicted by the Michaelis‐Menten equation; the I‐V relationship will remain linear. We find that Erev shifts appropriately for pHo 9.0‐6.0, excluding #1. Importantly, the I‐Vs are linear and exhibit only small GNBC decreases at low pHo, eliminating #4 & #5. In fact ‐‐ based on the experimentally determined Km(CO3=) = 4 μM (constant pHo 7.5 and 5% CO2)‐‐ these GNBC decreases at low pHo, and the increases at high pHo are smaller than expected. In conclusion, the major carbon substrate for NBCe1 must be either CO3= or NaCO3‐ and, extreme pHo paradoxically modulates NBCe1 to minimize changes in transport rate.

Grant Funding Source: Supported by NIH DK30344 and NS18400
Age‐related activation of the hepatic unfolded protein response and inflammasome are reduced by trehalose treatment (1115.2)
Authors: Pagliassotti M.J. , LaRocca T.J. , Wei Y. , Wang D. , Hudson W.M. , Zigler M.L. , Seals D.R.
Published: June 2021

The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging, pathological states and cell death. Quality control systems, such as the unfolded protein response (UPR), inflammasome and autophagy, mitigate the residence time of damaged proteins. In the present study, we have examined the UPR and inflammasome in the liver of young (Y; ~ 5 mo.) and old (O, ~29 mo.) mice (n=8/group) and the ability of trehalose, a compound linked to increased protein stability and autophagy, to counteract aging‐induced effects on these systems. When used, trehalose was provided for 4 wks in the drinking water (n=7/group). Livers from O mice were characterized by activation of the UPR and inflammasome and increased indices of liver injury. Mitofusin‐2 (Mfn‐2), a protein linked to protection against endoplasmic reticulum stress and UPR activation, was reduced in O vs. Y. Trehalose treatment reduced the activation of the UPR and inflammasome, and restored Mfn‐2 in O mice. It has been postulated that a collapse of proteostasis promotes many age‐associated pathologies. Trehalose appears to be an effective intervention to mitigate the activation of quality control systems that respond to disruption of proteostasis in the liver. We postulate that the age‐associated reduction in Mfn‐2 is critical to the activation of the UPR and inflammasome in the liver of old mice.

Grant Funding Source: NIH DK072017, AG013038, AG039210
Leptin receptor deletion from POMC neurons increases susceptibility to high fat diet‐induced obesity (1126.9)
Authors: Bell Balyssa , Harlan Shannon , Morgan Donald , Rahmouni Kamal
Published: June 2021

Leptin, an adipocyte derived hormone, acts in the central nervous system (CNS) to regulate energy homeostasis. Neurons in the hypothalamic arcuate nucleus such as proopiomelanocortin (POMC) neurons represent a major target of leptin to regulate metabolism. However, conditional deletion of the leptin receptor (LepR) from POMC neurons has been shown to result in only a mild obesity phenotype while global or CNS LepR knockout mice are severely obese. Here, we examined the susceptibility of mice lacking the LepR in POMC neurons to high fat feeding. Mice missing the LepR specifically in POMC neurons were generated by crossing LepRfl/fl with POMCCre. LepRfl/fl‐POMCCre mice and littermate controls were fed normal chow or high fat diet (HFD, containing 45% fat/kCal) from 4 to12 weeks of age. Consistent with previous reports, on normal chow, LepRbfl/fl‐POMCCre mice have a slight, but significantly (P<0.05), elevated body weight (31±1 g) relative to littermate controls (28±1 g). Strikingly, on HFD, LepRbfl/fl‐POMCCre mice exhibited an exaggerated increase in body weight (42±1 g) as compared to control littermates (31±1 g, P<0.05). Fat mass was also significantly (P<0.05) increased in HFD fed LepRbfl/fl‐POMCCre mice (3.4±0.1g) relative to control mice (1.9±0.2g). Our results provide direct genetic evidence that LepR in POMC neurons is required for normal adaptation against diet‐induced obesity.
Spontaneous essential hypertension in the non‐human primate Chlorocebus aethiops sabaeus, the African green monkey (1136.16)
Authors: Rhoads Megan , Beierschmitt Amy , Huggins Denise , Collett Jason , Osborn Jeffrey
Published: June 2021

Current models of essential hypertension have limited comparisons to humans due to differences in posture and periods of activity. The annual cycle of spontaneous hypertension in male Chlorocebus aethiops sabaeus was assessed in an outbred research colony. Using plethysmography, systolic (SBP) and diastolic blood pressures (DBP) of adult male African Green, or vervet, monkeys (n=163) were measured in May/June after ketamine sedation (~10‐15mg/kg). Blood pressure phenotypes were characterized as hypertensive (HT: SBP 蠅 140mmHg) or normotensive (NT: SBP 蠄 120mmHg). SBP and DBP identified 33% (n=53) HT (average SBP 169.04±24.19mmHg) and 41% NT (average SBP 101.32 ±13.28mmHg, n=66) animals. Borderline hypertension (BHT) was identified with an average SBP of 129.09±5.85mmHg (n=44). A randomized group of animals (n=51) were re‐measured in September/October. HT monkeys (n=21) decreased blood pressure from 174.14±27.48mmHg to 128.61±28.48mmHg (p<0.05). SBPs of NT (n=21) and BHT animals (n=9) were unchanged (p>0.05). A third of adult male vervet monkeys in this colony exhibit spontaneous hypertension with unique seasonal variation compared to NT or BHT animals. This model of hypertension in an Old World non‐human primate is highly translational to human essential hypertension and allows for new research directions in a novel genetically similar species.

Grant Funding Source: Supported by Behavioural Science Foundation
Multiple mechanisms of PAHs toxicity in Daphnia magna: effects of an AhR agonist and competitor on genes involved in toxicant pathways (1138.11)
Authors: Askounis Erin , Augustine Marianna , Lee Jason , Vulpe Chris
Published: June 2021

Understanding the mechanistic pathways underlying the toxicity of chemicals can provide more biologically relevant models to extrapolate adverse outcomes. Mechanistically based models should provide more efficient means to screen untested compounds and predict exposure to untested species. We evaluated the relationship between reproductive outcomes in Daphnia magna and expression of biomarkers after exposure to polyaromatic hydrocarbons (PAHs). These biomarkers include Daphnia magna homologs of ARNT (aryl hydrocarbon nuclear translator), CYP4 (Cytochrome P450 Family 4), COX (cyclooxygenase), retinoid‐X‐receptor (RXR), and juvenile hormone esterase (JHE). To assess physiological endpoints we monitored reproductive changes in a 14‐day chronic exposure. Exposure to benzo(a)pyrene resulted in altered reproduction and was correlated with increases in RXR and decreases in VTG and CYP4 genes. We then conducted exposures with a known cytochrome P450 inhibitor, PBO (piperonyl butoxide and phenanthrene. PBO alone induced ARNT, JHE, COX, RXR, and CYP4 more than phenanthrene alone. In combination with PBO, phenanthrene did not induce JHE, RXR, and downregulated phase I detoxification pathway genes ARNT and CYP4. CYP450 activity (ARNT) does not have a role in induction of developmental hormones (JHE). These results indicate that Daphnia toxicant control mechanisms are operating through multiple pathways that influence reproduction and survival.