bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Liquid Sugar Reduction on Behavioral and Brain Responses to Food Viewing
Authors: Crézé Camille , Bielser Marie‐Laure , Knebel Jean‐François , Campos Vanessa , Tappy Luc , Murray Micah , Toepel Ulrike
Published: June 2021

Obesity is increasingly considered as a brain disease in which homeostatic mechanisms regulating food intake are overridden by hedonic drives towards consumption of foods high in fat and sugar. How added sugars from sugar‐sweetened beverages (SSBs) impact hedonic drives to foods, and whether artificial sweeteners have specific effects of their own remains unknown.

We studied 14 high SSB consumers (8 men, 6 women) before and after 3 months of SSB replacement by artificially sweetened equivalents. Participants performed behavioral preference ratings on a 5‐point scale when presented with color images of solid foods differing in fat content (high‐ vs. low‐fat) and taste quality (savory vs. sweet). Moreover, the spatio‐temporal brain dynamics while viewing the food types were assessed using electroencephalography (EEG) and electrical neuroimaging analyses.

Behavioral results show a significantly greater appreciation of savory foods, but decreased appreciation of sweet foods post‐SSB replacement. Brain responses reveal diet‐induced modulations in particular to low‐fat foods in areas involved in food valuation and cognitive control over food intake. That is, the replacement of liquid calories substantially influences responsiveness to solid calories, likely explaining changes in food intake behavior. Whether these effects are due to sugar reduction or to artificial sweeteners per se remains to be investigated.

This work was supported by the Swiss National Science Foundation.
Trans Fatty Acids in Plasma, Serum, and Red Blood Cells
Authors: Kuiper Heather , Vesper Hubert
Published: June 2021

Trans‐fatty acids (TFA) intake was associated with increased risk of cardiovascular disease. However, little is known about TFA concentrations in human blood. Studies suggested that individuals with the same fatty acid (FA) intake may have different concentrations of regular FA in red blood cells (RBC), plasma, and serum due to a number of factors including genetics and lifestyle. This created the need to assess TFA concentrations in different blood specimens from people to obtain information about TFA exposure and metabolism. We investigated the TFA distribution in RBC, plasma, and serum using convenience samples from 66 adults. Samples were analyzed using an isotope dilution‐gas chromatography‐mass spectrometry method validated for the analysis of 28 FA, including the 4 major TFA: palmitelaidic acid, elaidic acid, vaccenic acid, and linoelaidic acid. The TFAs were detected in all samples, with median total TFA concentrations of 21.12 μM in RBC, 17.86 μM in plasma, and 20.88 μM in serum. The median percent of total TFA was similar for elaidic and vaccenic acids in all three matrices. The median percent of total TFA was 1/3 lower in the RBC than in the plasma and serum for palmitelaidic acid and half as high for linoelaidic acid in RBC as in plasma and serum. The differences in TFA distributions suggest differences in metabolism of individual TFA.
Potential Role of PYY in Skeletal Muscle Cells?
Authors: Gupta Diwakar , Roman Heather , Touhamy Samir , Lin Kevin , Thalacker‐Mercer Anna
Published: June 2021

PYY is characteristically considered an appetite hormone with identified roles in metabolism, inflammation, and proliferation. The expression of PYY and its receptors has been demonstrated in the skeletal muscle; however, the role of PYY in the skeletal muscle is unknown.

Objective

To investigate the role of PYY on proliferating and differentiating/differentiated skeletal myoblasts and to determine the effect of PYY on inflammatory and metabolic signaling pathways.

Methods

Expression of the PYY gene was determined in cultured murine C2C12 cells incubated in growth or differentiation media (GM and DM, respectively) using quantitative PCR. Additionally, changes in proteins of interest were determined from C2C12 cells that were cultured in DM for 96 h then switched to DM containing 0.1 mM palmitate for 72 h, followed by exposure to 500 pM murine PYY3‐36 for 6 h prior to lysis.

Results

PYY gene expression, which was identified in proliferating C2C12 cells, decreased in differentiating cells. Relative to myotubes that received only palmitate, the presence of PYY decreased phosphorylation of STAT3 (signal transducer and activator of transcription 3) serine‐727 (‐37%, p = 0.03) and tyrosine‐705 (‐33%, p = 0.01). There was no change in the total STAT3 protein, the ratio of phospho‐STAT3 to total STAT3, or NF‐kB‐p65 levels.

Conclusions

These results indicate that the PYY gene is not only detectable in mouse myoblasts, but it also changes with different physiological states (i.e. proliferating vs. differentiating). Additionally, PYY appears to play a role in skeletal muscle STAT3 signaling. Follow‐up research is necessary to confirm these results and identify downstream targets of PYY through the STAT3 pathway and functional outcomes. Support: NIDDK P30DK056336
The Effect of Mango Supplementation on Clinical Parameters of Pre‐Diabetic Individuals
Authors: Semkoff Jess , Evans Shirley , Janthachotikun Sawanya , Eldoumi Heba , Mahmood Maryam , Meister Maureen , Payton Mark , Peterson Sandra , Perkins‐Veazie Penelope , Clarke Stephen , Smith Brenda , Lucas Edralin
Published: June 2021

Diabetes (DM) is a leading cause of death in the United States due to the complications associated with hyperglycemia. Type 2 DM has increased in prevalence due to the rise in obesity. The pre‐diabetes (fasting blood glucose of 100‐125 mg/dl) state represents a critical point for intervention that may delay or prevent the development of DM. Previously, we have shown that mango supplementation lowered blood glucose in obese individuals. This study investigated the effects of freeze‐dried mango supplementation on clinical parameters of pre‐diabetic individuals. Mango fruit is rich in vitamins and minerals, fiber, and phenolic compounds, which possess hypoglycemic, anti‐inflammatory and antioxidant properties. In a cross‐over study design, pre‐diabetics (n=28) were randomly assigned to consume either freeze‐dried mango (10 g) or placebo supplement (isocaloric to the mango supplement) once daily for 12 weeks with a 3‐4 week wash‐out period between the two treatments. After 12 weeks supplementation, there were no significant differences in glycemic indices between mango and placebo treatment groups; however, a trend of decreased blood glucose and increased insulin levels were observed with mango supplementation. There were no significant differences in body composition or clinical parameters between groups. Unlike our earlier findings, chronic consumption of mango fruit has a modest effect in lowering blood glucose in pre‐diabetics.
β‐Cryptoxanthin and Its Cleavage Metabolite, 3‐OH‐β‐Apo‐10′‐Carotenal, can Inhibit LPS‐induced Inflammatory Responses in Human Bronchial Epithelial Cells
Authors: Hu Kang‐Quan , Mein Jonathan , Wang Xiang‐Dong
Published: June 2021

We have previously demonstrated that supplementation with the xanthophyll β‐cryptoxanthin (BCX)suppresses smoke/nicotine induced‐inflammation, emphysema, and lung tumorigenesis in animal models, which is likely to be independent of its provitamin A activity. These studies raised the question as to whether the protective effects of BCX result from the intact molecule or from its metabolites, independent of vitamin A. In the present study, we examined the effects of BCX and its cleavage metabolite by β‐carotene‐9′,10′‐oxygenase, 3‐OH‐β‐apo‐10′‐carotenal (3OH‐BA10C), against lipopolysaccharide (LPS)‐induced inflammatory responses of human bronchial epithelial cell line BEAS‐2B in vitro. Results showed that pre‐treatments of either BCX or 3OH‐BA10C in BEAS‐2B cells up‐regulated sirtuin 1 (SIRT1, a NAD+‐dependent protein deacetylase) protein expression and inhibited the LPS‐induced mRNA expressions of IL‐6 and TNF‐α in a dose‐dependent manner (1 to 4 μM concentrations). While these effects were associated with reduced AKT phosphorylation, BCX, not 3‐OH‐BA10C, up‐regulated IκB‐α (inhibitor of NF‐κB) protein without affecting its phosphorylation in BEAS‐2B cells. In contrast, 3OH‐BA10C induced the expression of hemo oxygenase‐1 (HO‐1) at both mRNA and protein levels in both dose‐ and time‐dependent manners. Taken together, these data demonstrate that both BCX and 3OH‐BA10C, at comparable doses, attenuate LPS‐induced inflammatory responses via differential molecular mechanisms, indicating thatthese biological activities could be due to action of their own molecule. Supported by the NIH grant CA176256
Effects of consuming an iron‐biofortified bean on behavioral and electrophysiological measures of attention
Authors: Wenger M , Rhoten S , Scott S , Murray‐Kolb L , Lung'aho M , Gahutu J‐B , Haas J
Published: June 2021

Iron deficiency (ID) can produce deficits in cognition and brain dynamics, and repletion may reverse these changes. We conducted a double‐blind efficacy study involving healthy female students (n = 239) screened for low iron status. Participants were randomized to groups that consumed either a high‐iron‐biofortified (BB) or a normal‐iron bean (CN) for 20 weeks. A subsample (n = 53) performed cognitive tasks while concurrent EEG was acquired at baseline (BL), endline (EL), and at one of eight random time‐points in between. Cognitive tasks included (a) a measure of perceptual speed (simple reaction time, SRT); (b) a test of inhibitory control (go/no‐go, GNG); and (c) an assessment of three aspects of attention (attentional network task, ANT). Small but significant improvements from BL to EL were observed for hemoglobin, ferritin, and total body iron, with larger improvements for the BB vs CN group. Behaviorally, there were no improvements in the SRT, possibly reflecting a floor‐effect. Significant condition (BB, CN) x time interactions were obtained for the behavioral measures from the GNG and ANT. The EEG data revealed significant differential increases for the BB vs CN group in the amplitudes of two components (N1 and P2), and normalized spectral power in two frequency ranges (alpha and gamma) that index aspects of attention in all three tasks. The results suggest that consuming BB may benefit behavior and brain function.
Influence of açaí supplementation in cardiac remodeling after myocardium infarction in rats
Authors: Figueiredo Amanda , Polegato Bertha , Cardoso Ana Carolina , Candido Renata , Rafacho Bruna Paola , Dos Santos Priscila , Batista Diego , Gonçalves Andréa , Okoshi Katashi , Gaiola Paula , Zornoff Leonardo , Minicucci Marcos , Paiva Sergio Alberto
Published: June 2021

One of the determining factors for cardiac remodeling after myocardial infarction (MI) is oxidative stress. Fruits, such as açaí, which modulate oxidative stress, may attenuate cardiac. (Vissotto, 2013)

Objective

analyze the influence of açaí supplementation in chow on the cardiac remodeling after MI.

Material and methods

After MI, male rats Wistar weighing from 200 g were allocated into 6 groups: 1) Sham group fed with standard chow (SA0, n=5); 2) Sham group fed with standard chow with 2% açaí (SA2, n=5); 3) Sham group fed with standard chow with 5% açaí (SA5, n=5); 4) MI standard chow (IA0, n=5); 5) MI and chow with 2% açaí (IA2, n=7); 6) MI and chow with 5% açaí (IA5, n=7). After 3 months of supplementation, echocardiography study was conducted. Two‐way ANOVA was used and values are expressed as mean±SE.

Results

After 3 months, the Infarct size did not differ among the MI groups. In MI rats, it was observed an increase in the left ventricular diastolic diameter, left ventricular systolic diameter, left ventricle posterior wall thickness, left atrial diameter (LA)/ aortic diameter (Ao) and a reduction in the fractional area change and A mitral wave. The addition of açaí pulp in the diet reduceLA (LA, LA/Ao, LA/BW), increase A mitral wave andreduce the E mitral wave and E/A. No difference was observed between IA2 and IA5.

Conclusion

açaí supplementation modulates cardiac remodeling induced by MI, improving the diastolic function.
Dietary plant lectins affect lifespan and dopaminergic neurons in Ceanorhabditis elegans model organism
Authors: Wang Mingming , Hockensmith Adam , Laine Roger , Keller Jeffrey , Heymsfield Steven , Gao Chenfei , Nguyen Jenny , Zheng Jolene
Published: June 2021

Braak proposes that an “unknown pathogen” penetrates the gastrointestinal and nasal wall, and by retrograde/anterograde transport, enters the central nervous system. Dietary plants' lectins can trans‐synaptically transport into the nervous system suggesting a possibility of acting as an etiologic agent and/or chaperone for viruses/toxin(s) to induce PD. A TRITC‐lectins' library is conducted in C.elegans (egIs1 [dat‐1p::GFP]) which were maintained in liquid culturof in 96‐well plates (20°C), with lectins with or without additional hapten. The average lifespan was 18 days in the control. Cytisus scoparius lectin showed a dose‐dependent “biphasic” relationship mildly increased the lifespan at 2‐6µg/ml, which was reversed at 20µg/ml, antagonized byGaINAc (20mM). Lectins from Amaranthus caudatus (ACA), Galanthus nivalis (GNA) and Euonymuseuropaeus (EEA) decreased lifespan dose‐dependently (2‐20µg/ml, P<0.05). Confocal microscopy revealed that TRITC‐ACA or ‐EEA co‐localized with GFP‐DAergic neurons. These data indicated that dietary plant lectins were transported from intestine to the DAergic neurons and negatively affected lifespan in C. elegans, which supports the hypothesis of an “unknown pathogen” in Parkinson's disease.
Chronic Intake of Wild Blueberry Extract and Powder Maintains Episodic‐Like and Working Memory During Normal Aging in Mice
Authors: Fromentin Emilie , Krazem Ali , Henkouss Nadia , Roller Marc , Beracochea Daniel
Published: June 2021

The number of Americans older than 65 years old is projected to more than double in the next 40 years. Cognitive changes associated to aging can affect adult's day‐to‐day functioning. Early memory changes include decline in both working and episodic memory. The aims of the present study were to determine whether chronic (up to 75 days) daily administration of wild blueberry (Vaccinium angustifolium) extract or a wild blueberry whole fruit powder would help prevent memory failure associated with aging, in tasks involving various forms of memory. Both blueberry ingredients were used in a study comparing young mice (6‐month old) to aged mice (18‐month old). At this age, mice exhibit memory declines due to aging, which are exacerbated first by losses in working and contextual (episodic‐like) memory. Contextual (Episodic‐like) memory was evaluated using the Contextual Serial Discrimination test (CSD). Working and spatial memory were evaluated using the Morris‐Water maze test and the Sequential alternation test. Statistical analysis was performed using an ANOVA with Bonferroni post‐hoc test. Supplementation with wild blueberry powder and blueberry extract resulted in significant maintenance of contextual memory, while untreated older mice experienced a decline in such memory. Only the wild blueberry powder significantly contributed to the maintenance of spatial and working memory versus untreated older mice. These improvements of cognitive performance may be related to brain oxidative status, acetylcholinesterase activity, neuroprotection, attenuation of immunoreactivity.
Thrombomodulin Inhibits Tissue factor Mediated Thrombin Generation in blood and Prothrombin Complex Concentrates
Authors: Fareed Jawed , Hoppensteadt Debra , Syed Daneyal , Kahn Daniel , Mosier Michael , Kaul Inder
Published: June 2021

A recombinant thrombomodulin (Recomodulin, Asahai Kasei, Tokyo, Japan) is currently used for the management of DIC and related disorders. Beside inhibiting thrombin and forming complexes with this enzyme, Recomodulin (RM) mediates its therapeutic effects involving modulation of inflammatory processes and thrombogenesis. The purpose of this study is to determine the effect of RM on thrombin generation in plasma and prothrombin complex concentrates.

RM (2.02 mg/ampule) was commercially obtained. Working solutions of buffered RM were prepared at 100ug/ml. Tissue factor mediated generation of thrombin and its inhibition in normal human plasma (NHP) (n=25), platelet rich plasma (PRP) (n=25) and various prothrombin complex concentrates such as Beriplex, Cofact, Feiba, Konyne, Octaplex, Preconativ, Profilnin, Prothromplex ( 1u/ml) were investigated utilizing a fluorometric thrombokinetics method. The effect of RM on thrombin mediated fibrinokinetics was also measured using an optical method.

RM produced a concentration dependent inhibition of thrombin generation in all systems. In the NHP the IC50 was 0.29 + 0.06 ug/ml in contrast to PRP where the IC50 was 1.99 + 0.31 ug/ml. The inhibition of thrombin generation in various PCCs was also concentration and product dependent. Marked inhibition was noted in Preconativ, Prothromplex and Beriplex. RM also inhibited the fibrin formation in both the PRP and NHP system in a differential manner.

These results suggest that beside the direct inhibition of thrombin, RM is capable of inhibiting thrombin generation in a matrix dependent fashion and is also capable of modulating fibrin formation as evident by fibrinokinetic studies.
The Intimal Alteration of Human Varicose Veins Is Associated with Oxidative Stress and Cyclooxygenase‐2 Dependent Mechanisms
Authors: Velasco‐Martin Juan , Aguado Andrea , Mendez Iago , España‐Caparros Gabriel , Salaices Mercedes , Regadera Javier , Briones Ana
Published: June 2021

Chronic venous insufficiency (CVI) determinates high morbidity. Main varicose histological lesions include intimal fibrosis, luminal dilation and progressive vascular wall thickening. Plasma biomarkers of oxidative stress are increased in CVI patients. We evaluated histological changes and local inflammatory profile, particularly oxidative stress and prostanoids, in varicose saphenous veins (SV).

10 distal and proximal segments of SV from patients with CVI were studied. In intimal layer, expression of smooth muscle actin (SMA), collagen I/III, and elastic fibers distribution were quantified by ImageJ sofware. NADPH oxidase subunit 4 (NOX‐4), cyclooxygenase‐2 (COX‐2), microsomal prostaglandin E synthase (mPGES‐1),MAC3, SMA, elastin, collagen I/III were studied by RT‐PCR. H2O2 production and NADPH oxidase activity were evaluated by commercial kits.

Histological analysis showed higher intimal expression of SMA, collagen I/III and elastic fibers in proximal segments compared to distal segments. The progressive intimal vein thickness is inversely related to the ratio of SMA, collagen I/III and elastic fibers respect to intimal area. mRNA expression of SMA, collagen I/III, elastin, NOX‐4 and COX‐2 but not of mPGES‐1 or MAC3, H2O2 production and NADPH oxidase activity are upregulated at proximal varicose segments compared to distal segments. Our results suggest that oxidative stress and COX‐2 derived prostanoids might be implicated in the initial stages of the intimal fibrosis observed in varicose SV. Supported by PI13/01488 and SAF2012‐36400.
Activating β‐catenin Mutations And AKT Synergize To Promote Lipogenic Liver Tumors In Mice
Authors: Tao Junyan , Li Xiaolei , Singh Sucha , Chen Xin , Monga Satdarshan
Published: June 2021

AKT/mTOR and Wnt/β‐catenin cascades are frequently deregulated in human tumors. Here, we establish a novel mouse model by co‐expression of active form of AKT (myr‐AKT) and β‐catenin mutants (S33Y, S45Y) in mouse liver using the sleeping beauty transposon/transposase delivered via hydrodynamic tail vein injection. The mice showed large liver tumors at 14‐15 weeks post injection for the AKT/S45Y‐β‐catenin group, and 16 weeks for the AKT/S33Y‐β‐catenin group. All tumors displayed notable lipid accumulation in hepatocytes. Activation of downstream target genes of Akt and β‐catenin were found in both models and included Raptor, Rictor, mitochondrial related genes PKM2 and Myc, Hippo/Yap/TAZ and Glutamine synthetase. To demonstrate their relative contribution in the HCC model, we sub‐cloned shRNA against various downstream targets and co‐delivered with AKT and β‐catenin mutants by hydrodynamic tail vein injection. Suppression of key target genes notably affected tumorigenesis in this model revealing their unique and specific biological functions. Thus, while differences in β‐catenin mutations can lead to differing extents of β‐catenin activation, b‐catenin does cooperate with AKT to induce HCC with lipogenic phenotype. Also several downstream targets of AKT/mTOR and β‐catenin pathways play key roles in HCC development.
Computational Modeling of a Cytokine Interaction Network in Microglia
Authors: Anderson Warren , Makadia Hiren , Schwaber James , Vadigepalli Raj
Published: June 2021

Neuroinflammation due to glial activation has been linked to many CNS conditions. To examine the mechanistic underpinnings of how intercellular cytokine interactions coordinate the neuroinflammatory milieu, we employed a dynamic modeling approach. We developed a microglial cytokine interaction model using differential equations. The model fit to published data from LPS‐stimulated cultured microglia demonstrates that the model captures the dynamics of cytokine regulatory interactions. Results from a variance‐based parametric sensitivity analysis suggest that TNFα is highly sensitive to negative feedback from TGFß and IL‐10. Based on this analysis, we examined the relative roles of these anti‐inflammatory cytokines in controlling TNFα levels in the contexts of adaptation and tolerance to LPS stimuli. Simulations examining adaptation of the TNFα response to LPS indicate that TGFß controls TNFα adaptation by modulating the steady‐state response to LPS, with negligible effects on the peak TNFα response. In contrast, IL‐10 primarily controlled the TNFα peak amplitude following LPS with less effect on adaptation. Consistent with experimental data, the model simulations also showed tolerance in the TNFα response to repeated doses of LPS. A simulated IL‐10 knockout (KO) phenotype resulted in enhanced TNFα tolerance whereas simulated TGFß KO hyper‐sensitized the TNFα response to repeated LPS stimuli. The contrasting effects of TGFß versus IL‐10 occurred because the IL‐10 KO enhanced the initial TNFα peak response, which resulted in subsequent TGFß activation and robust negative feedback, thereby rendering TNFα unresponsive to a second LPS stimulus.
Selective Antagonism of mGlu5 Alters Sleep‐wake and Spectral EEG and Ameliorates Behavioral Abnormalities in a Rodent Model of Traumatic Stress
Authors: Nedelcovych Michael , Gould Robert , Gong Xuewen , Felts Andrew , Grannan Michael , Thompson Analisa , Ivarsson Magnus , Emmitte Kyle , Lindsley Craig , Conn Jeffrey , Jones Carrie
Published: June 2021

Traumatic stress induces state‐dependent alterations in cortical network activity as measured by sleep‐wake and quantitative electroencephalography (qEEG) that may predict the onset or severity of chronic post‐traumatic stress disorder (PTSD) symptoms. Activation of metabotropic glutamate receptor 5 (mGlu5) promotes fear memory consolidation, and may contribute to sleep disturbances after trauma. The single prolonged stress (SPS) rodent model of traumatic stress recapitulates many physiological and behavioral correlates of the human disorder, but its effect on sleep‐wake and qEEG spectral power has not been examined.

Male Sprague‐Dawley rats were implanted with telemetric EEG electrodes continuously recording cortical activity before and after SPS. VU0409106, a selective mGlu5 negative allosteric modulator (NAM) was administered after SPS to assess blockade or reversal of SPS‐induced sleep‐wake and qEEG disturbances.

SPS caused persistent PTSD‐like alterations in rapid eye movement (REM) sleep and non‐REM (NREM) sleep as well as changes in state‐dependent spectral power, such as an increase in waking beta (18‐30Hz) power, and a decrease in NREM delta (0.5‐4Hz) power indicative of hyperarousal and poor sleep quality. VU0409106 modulated SPS‐induced sleep‐wake architecture changes and qEEG spectral power alterations and ameliorated accompanying anxiety‐like behaviors.
Regulator of G protein Signaling 14 (RGS14) Interacts with 14‐3‐3 via an H‐Ras and Gαi‐dependent Mechanism
Authors: Gerber Kyle , Seyfried Nicholas , Hepler John
Published: June 2021

RGS14 is a multifunctional protein scaffold that is known to interact with active Gαi/o via its RGS domain, Raf and active H‐Ras‐GTP via its R1 Ras binding domain (RBD), and inactive Gαi 1/3 via its G protein regulatory (GPR; also known as GoLoco) domain. RGS14 has been shown to suppress long term potentiation (LTP) in the CA2 region of the hippocampus, thereby regulating hippocampal‐based learning and memory. The 14‐3‐3 family of proteins has recently been shown to be necessary for hippocampal LTP and associative learning and memory. Here we show evidence for an H‐Ras and Gαi‐dependent interaction between RGS14 and 14‐3‐3. Following overexpression of FLAG‐RGS14 and 14‐3‐3γ in HEK293T cells and pull‐down of FLAG‐RGS14, we observed an interaction between RGS14 and 14‐3‐3γ only in the presence of Gαi and/or the constitutively active form of H‐Ras(G12V). This interaction appears to occur within the R1 domain of RGS14, and is corroborated by mass spectrometry analysis of proteins recovered from intact cells. RGS14 interaction with 14‐3‐3γ does not appear to be due to direct binding of Gαi or H‐Ras(G12V) to RGS14. Ongoing studies will further elucidate the nature and physiological function of this interaction between RGS14 and 14‐3‐3γ, which may give insight into the functions of both RGS14 as well as 14‐3‐3 in their roles in modulating synaptic plasticity in the hippocampus. Studies supported by R01‐NS037112‐15 (JRH) and T32‐GM008602 (KJG).
PI4P Hydrolysis Represents a General Mechanism for DAG Generation and PKC/PKD Activation
Authors: Gil de Rubio Rafael , Malik Sundeep , Wang Liwei , Smrcka Alan
Published: June 2021

Receptor stimulated hydrolysis of phosphatidylinositol 4,5‐bisphosphate (PIP­2­) is critical for many physiological processes, such as neurotransmission and proliferation. Recently our laboratory identified a novel pathway in neonatal rat ventricular cardiac myocytes (NRVMs), where agonist stimulation triggers PLC dependent hydrolysis of the PIP­2­ precursor phosphatidylinositol 4‐phosphate (PI4P) leading to DAG production, nuclear PKD activation and cardiac hypertrophy. We have seen that this newly identified pathway exists in other cell types as well, such as Mouse Embryo Fibroblasts (MEFs) and PANC‐1 cells. To determine if PLC dependent PI4P hydrolysis represents a general mechanism for DAG generation and PKC/PKD activation we examined PI4P depletion and PKD activation in other cell types. In our studies we used a PI 4‐kinase inhibitor, phenylarsine oxide (PAO) or expression of a Golgi specific 4‐phosphatase, Sac1‐K2A (Sac1) to deplete PI4P. PAO treatment or Sac1 expression in NRVMs and MEFs, showed a dramatic reduction of inositol phosphate accumulation after endothelin‐1 (ET‐1) stimulation. PAO treatment completely blocked ET‐1 dependent global PKD activation in NRVMs and MEFs but not neurotensin (NT) dependent PKD activation in PANC‐1 cells. Sac1 expression showed reduced ET‐1 dependent global PKD activation in NRVMs and MEFs but did not change the NT dependent PKD activation in PANC‐1 cells. These results suggest that though Golgi PI4P hydrolysis occurs in multiple cell types, NRVMs and MEFs use PI4P hydrolysis as a source for DAG to promote PKD activation. This shows that PI4P hydrolysis occurs upon receptor stimulation and provides a novel signaling mechanism with numerous implications in physiology and disease.
Epac2 Mediates Cyclic AMP‐dependent Growth Arrest in NS‐1 cells Independently of Rap1
Authors: Eiden Lee , Eiden Maribeth , Emery Andrew
Published: June 2021

We have investigated the upstream activators, and downstream effectors, of Epac2 signaling in the NS‐1 pheochromocytoma cell line in which three cyclic AMP sensors, Epac, PKA and NCS/Rapgef2 mediate distinct cellular outputs: p38‐dependent growth arrest; CREB‐dependent cell survival; and ERK‐dependent neuritogenesis, respectively (Emery et al., Sci. Sig. 6: ra51, 2013; Emery et al., JBC 289: 10126, 2014). Based on the growth arrest assay in NS‐1 cells, the Gs‐coupled GPCRs ADCYAP1R1, ADRB1, ADRB2 and ADORA2 all couple efficiently to Epac2 in these cells. We have previously shown that cAMP‐dependent growth arrest in these cells a) requires Epac2 (using siRNA and appropriate controls (Emery et al., JBC 289: 10126, 2014) and b) does not require Rap1 activation (ibid). Alternative candidates for mediation of growth arrest by the GPCR‐Gs‐cAMP‐Epac2 pathway include Rap2, Rit1, Rin, Rac and perhaps other small GTPase effector proteins for which Epac2 could conceivably provide catalysis of activation (GDP release or GDP/GTP exchange). Despite the observation that neither Epac1 nor Epac2 can function as a Rit‐GEF in vitro (Shi et al., MCB 26: 9136, 2006), quantitative expression analysis is consistent with Rit1 as the major candidate for mediating cAMP‐ and Epac2‐dependent activation of p38 leading to growth arrest in these cells, perhaps requiring an additional partner besides Epac2 for Rit1 activation in the pathway GPCR‐>Gs‐>AC‐>cAMP‐>Epac2‐>Rit1‐>MEKK‐>MEK‐>p38‐growth arrest. This pathway is distinctly parcellated from the GPCR‐>Gs‐>AC‐>cAMP‐>PKA‐>CREB and GPCR‐>Gs‐>AC‐>cAMP‐>NCS/Rapgef2‐>B‐Raf‐>MEK‐>ERK pathways which mediate cell survival and neuritogenesis, respectively, in NS‐1 cells.
PAC1‐Gs Coupling Through AC6 Mediates NCS/Rapgef2‐dependent ERK Activation in Neuroendocrine Cells
Authors: Emery Andrew , Eiden Maribeth , Eiden Lee
Published: June 2021

The neuritogenic cAMP sensor (NCS), the protein product of the Rapgef2 gene, couples GPCR‐Gs‐dependent elevation of cAMP to ERK activation required for neuritogenesis, while additional aspects of cAMP‐dependent cell differentiation are mediated through PKA (pro‐survival effects) and Epac2 (growth arrest) (Emery et al., JBC 289: 10126, 2014). Functional full‐length Rapgef2 is expressed in cell lines of neuroendocrine lineage, but not in several non‐neuroendocrine cell lines commonly used for high‐throughput GPCR ligand and antagonist screening (Emery et al., Sci. Sig. 6: ra51, 2013). In a series of NS‐1 cell sublines into which Gs‐coupled GPCRs were introduced as single‐copy genes under the control of the CMV promoter, some (e.g. ADCYAP1R1, ADBR1, DRD1), but not others (ADBR2, ADORA2A), engage NCS/Rapgef2 and induce neuritogenesis when stimulated with the appropriate ligand. All are coupled to adenylate cyclase and cause stimulation of Epac and PKA. To determine the mechanism of coupling specificity between GPCRs and NCS/Rapgef2, we are evaluating the membranous adenylate cyclase (AC) specificity for NCS/Rapgef2 coupling. In PC12 cells, which express ACs 3, 4, 6, 7, and 9, PACAP‐initiated signaling through ADCYAP1R1 (cAMP elevation, ERK phosphorylation, and neuritogenesis) is greatly attenuated by knockdown of AC6, but not AC7, achieved by stable lentiviral‐mediated shRNA expression, with no significant effects of either knock‐down on forskolin‐induced downstream cAMP‐dependent signaling events (ERK activation and neuritogenesis). These data suggest that the ADCYAP1R1 (PAC1) receptor is preferentially coupled to AC6, and this AC preference is linked to engagement of NCS/Rapgef2, ERK activation, and neuritogenesis.
Encapsulation Technology for Increased Portability of Cell‐based Biosensors
Authors: Curtis Theresa , Puoplo Nicholas , Plante Eric , Tison Christopher , Butler Blaine
Published: June 2021

Mammalian cell‐based biosensors (CBBs) have become a powerful way to detect pathogens, toxins and toxicants. CBBs can serve as a sentinel for a broad range of agents, including unknown or complex combinations of agents, and report a physiologically relevant endpoint. Despite CBBs advantages over conventional detection methods, they suffer from poor efficacy outside of the laboratory environment because cells are sensitive to changes in their environment including pH, temperature, and media composition. Advances in cell‐encapsulating hydrogels may solve some of the challenges with transitioning CBBs into commercial products. In collaboration with Luna Innovations Incorporated, sol‐gels were investigated for their cell stabilization activity. A well‐developed water toxicant sensor based on impedance sensing of confluent cell monolayers after chemical challenge was used to evaluate the sol gels. A variety of sol‐gel formulations and application protocols were evaluated to stabilize a confluent cell layer adherent to gold electrodes which is the sensing element of the impedance CBB. The data show that a saccharide modified sol‐gel was able to stabilize the impedance of a confluent layer of mammalian cells on the sensing platform for 50 days at room temperature with no medium replacement, whereas non‐encapsulated cells quickly died in this uncontrolled environment. Furthermore, encapsulated cells were still able to detect toxicants by exhibiting a decrease in impedance after treatment. These findings show that cell‐encapsulation may allow CBBs to transition from the laboratory into viable products. SBIR with ERDC‐CERL, #W9132T‐14‐C‐0016
FP Prostanoid Receptor Mediated Up‐regulation of Hypoxia‐Inducible Factor‐1α through Activation of Mitogen‐Activated Protein Kinase and Reactive Oxygen Species Signaling Pathways
Authors: Sanchez Christopher , Xu Wei , Chou Chih‐Ling , Regan John
Published: June 2021

The FP prostanoid receptor is in the family of G‐protein‐coupled receptors and is known to activate calcium and inositol phosphate signaling via coupling to Gq . It's complementary ligand is prostaglandin‐F2α (PGF2α). In human embryonic kidney (HEK) cells expressing the human FP receptor, we show that PGF2α stimulation of the FP receptor upregulates the expression of hypoxia‐inducible factor‐1α (HIF‐1α), under normoxic conditions, by activation of the mitogen activated protein kinase (MAPK) and reactive oxygen species (ROS) signaling pathways. Co‐expression of dominant negative Nurr1 (DN‐Nurr1) diminished the PGF2α stimulated upegulation of HIF‐1α expression. HIF‐1α is widely characterized in the literature as a tumor growth promoter and is often up‐regulated in cancer. Our findings provide a potential mechanism by which PGF2α could‐upregulate the expression of HIF‐1α through Nurr1 activation.
The Protective Role of Glutaredoxin‐1 in Endothelial Barrier Regulation and Atherosclerosis
Authors: Han Jingyan , Cohen Richard , Matsui Reiko
Published: June 2021

Vascular oxidative stress is widely invoked as an important pathogenic mechanism for atherosclerosis (AS). Protein S‐glutathionylation (P‐SSG), the prevalent post‐translational oxidative modification, is emerging as a critical redox signaling mechanism in cardiovascular diseases. However, its role in AS is not clear yet. P‐SSG can be removed by glutaredoxin‐1 (Glrx), a thiol‐transferase which plays a pivotal role in redox signaling. This study is aimed to investigate the role of P‐SSG in high fat‐induced endothelial barrier dysfunction and AS through genetic manipulation of Glrx in human aortic endothelial cell (HAEC) and mice. Overexpression of Glrx attenuates high palmitate high glucose (HPHG)‐induced reversible oxidation of proteins and hyper‐permeability of HAEC monolayer to dextran. Consistently, high fat diet‐induced aortic vessel leakage indexed by dye extravasation is markedly decreased in Glrx transgenic mice when compared with WT mice. Overexpression of hGlrx‐1 in HAECs stimulates cortical actin filament formation under both basal and HPHG challenge conditions. Employing biotin‐switch assays, we showed that Glrx up‐regulation diminished HPHG‐induced oxidative modification of actin and the RhoGTPases, Rac‐1. More importantly, Glrx‐1 corrected HPHG‐induced Rac1 inactivation, a key mechanism of impaired endothelial barrier integrity. Oxidative stress‐induced endothelial barrier dysfunction is an initial step of AS, we thus assessed the high fat diet‐induced AS lesions by en face Oil red O staining in Glrx‐1 transgenic mice with ApoE deficiency background, and found that Glrx‐1 up‐regulation significantly attenuates AS lesion formation. These results suggest a causal role of P‐SSG in the pathogenesis of AS and that Glrx may have therapeutic potential for AS.
Gender Difference in Coronary Endothelial Dysfunction in ApoE Knockout Mouse Atherosclerosis Model: Role of NO and A2A Adenosine Receptor
Authors: Zhou Xueping , Teng Bunyen , Mustafa S.
Published: June 2021

Gender plays an important role in the pathophysiology of cardiovascular diseases. Coronary endothelial dysfunction is a critical stage in the pathogenesis of artherosclerosis. This study aims to investigate how gender impacts the mechanisms of coronary flow regulation in apolipoprotein E‐knockout (ApoE KO) mouse model of artherosclerosis. ApoE KO mice fed with high fat diet for 12 weeks from the age of 8 weeks were used. Coronary flow (CF) of isolated hearts was measured using Langendorff system. Coronary reactive hyperemia (RH) and A23187‐induced NO‐dependent vasodilation were significantly reduced in female (by ~28% and 48%, respectively, p<0.05) but not in male ApoE KO mice. However, sodium nitroprusside (SNP, 10‐5M)‐induced vasodilation was comparable in ApoE KO vs. WT mice in both genders. L‐NAME (100 μM) inhibited the baseline flow and coronary RH to a lesser extent in ApoE KO (by ~18.8% and 31%, respectively) than WT mice (by ~ 29.5% and 59%, respectively, p<0.05), and was less effective in inhibiting coronary RH in female ApoE KO mice (17% vs. 45% compared with male, p<0.05). Inversely, SCH58261 (a selective A2A AR antagonist, 1 μM) inhibited the baseline flow and RH to a higher extent in ApoE KO vs. WT mice, but had similar effect in both genders. Immunofluorescent staining showed a similar extent of A2A AR up‐regulation in coronary arteries of ApoE KO female vs. male animals. Our results suggest that a more severed NO‐dependent endothelial dysfunction occurs in female than male ApoE KO mice and A2A AR up‐regulation serves as a compensatory mechanism in counteracting the decreased NO‐dependent endothelial dysfunction during artherosclerosis (supported by HL027339 & HL094447).
Silibinin Inhibits Glioblastoma Cell Proliferation via Akt/S6/4EBP1 and Stat3 Signaling Pathways In Vitro
Authors: Us Ilkay , Benzeroual Kenza
Published: June 2021

Glioblastoma is the most common primary and lethal brain tumor in humans. These malignant astrocytic tumors exhibit high proliferation rates and acquire resistance against many therapeutic regimens. Despite aggressive treatment including surgery, radiation, and chemotherapy, the median survival time of 12‐15 months after diagnosis has remained unchanged.Thus, new effective strategies for controlling glioblastoma are required. Because glioblastoma cells like any cancer cells avoid differentiation and apoptosis, the induction of differentiation and apoptosis in glioblastoma cells may be considered as a potential treatment strategy. Silibinin, a natural polyphenolic flavonoid, is a major bioactive component of silymarin, which is isolated from the plant milk thistle. Recent studies have indicated that silibinin has anticancer activities in various cancers including prostate cancer in both in vitro and in vivo models. The present study investigated the effect of silibinin on glioblastoma cell proliferation in vitro. U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. The expression and activity of AKT, S6K, 4EBP1, and STAT3 proteins were measured by Western blot analysis. Silibinin induced cell death as well as inhibited proliferation in a dose‐ and time‐dependent manner. Accordingly, levels of biologically active phospho‐AKT, phospho‐S6K, phospho‐4EBP1 and phospho‐STAT3 were decreased in a dose‐ and time‐dependent manner. This study demonstrates that silibinin bears an anti‐oncogenic property via inhibition of PI3K/Akt and STAT pathways against GBM cells in vitro. Thus, this study promises a great potential agent for Glioblastoma. Study supported by the science research funds of AMS College of Pharmacy, LIU, NY
Vascular Endothelial ACE Deletion does not Prevent 20‐HETE‐dependent Vascular Remodeling
Authors: Shkolnik Brian , Garcia Victor , Bernstein Kenneth , Falck John , Schwartzman Michal
Published: June 2021

In vivo, 20‐HETE‐mediated hypertension in rodents is associated with increased vascular ACE expression and circulating Ang II levels. Administration of an ACE inhibitor or Ang II receptor blocker attenuates 20‐HETE‐mediated increases in blood pressure. ACE3/3 mice lack vascular endothelial ACE, exhibit attenuated ACE expression in the kidneys and display normal ACE and circulating Ang II levels in the plasma. Administration of a 21 day release pellet of 5α‐dihydrotestosterone (DHT), a known inducer of 20‐HETE production, increased systolic blood pressure significantly in age‐matched wild type (WT) mice (148±4 vs 99±2 mmHg) as compared to ACE3/3 mice (125±1 vs 99±1 mmHg) on day 14 and was maintained till day 21. DHT treatment increased preglomerular microvessel (PGMV) remodeling in both WT and ACE3/3 mice; however, remodeling was attenuated in the ACE3/3 mice (media thickness, 15.83±1.11 vs 9.80±0.56 μm) as opposed to the WT mice (22.17±0.92 vs 9.68±0.76 μm). Concurrent treatment with DHT and 20‐HEDGE, a 20‐HETE antagonist, prevented the increase in blood pressure as well as the changes in vascular remodeling in WT and ACE3/3 mice. Mice treated with DHT and losartan have a normalized blood pressure throughout the 21 days yet microvascular remodeling was not prevented in WT and ACE3/3 mice (media thickness, 14.75±0.75 vs 15.30±1.21 μm, respectively). These results suggest that vascular endothelial ACE/AngII contribute, in part, to 20‐HETE‐mediated microvascular remodeling in hypertension and that 20‐HETE have effects on microvascular remodeling independent of its action on ACE/AngII in the vascular endothelium.
Soluble Junctional Adhesion Molecule‐B Inhibits Angiogenesis In Vitro
Authors: Carey Morgan , Pickworth Courtney , Tsou Pei‐Suen , Imhof Beat , Koch Alisa , Rabquer Bradley
Published: June 2021

Angiogenesis is a significant part of physiological processes such as healing, reproduction and development. It also plays a major role in pathologic processes including rheumatoid arthritis and tumor growth. Angiogenesis is controlled by a balance of pro‐ and anti‐angiogenic mediators. To date, there are many more known pro‐angiogenic factors, including vascular endothelial growth factor (VEGF). Junctional adhesion molecules (JAMs) are a newly described family of adhesion molecules that have been shown to play roles in inflammation and angiogenesis. Soluble JAM‐C (sJAM‐C) is a pro‐angiogenic mediator. Recently, inhibition of JAM‐B has been slow to reduce tumor volume and vascularity. In addition, our previous work has shown that sJAM‐B inhibits basic fibroblast growth factor induced angiogenesis. Based on these findings, we hypothesized that sJAM‐B is a broad based anti‐angiogenic mediator capable of inhibiting VEGF and sJAM‐C induced angiogenesis. To test this hypothesis, we performed in vitro angiogenesis experiments using human microvascular endothelial cells (HMVEC). We found that sJAM‐B decreased sJAM‐C induced HMVEC chemotaxis (n=8 replicates). In addition, sJAM‐B significantly inhibited sJAM‐C induced HMVEC tube formation on Matrigel (p<0.05, n=9). Moreover, sJAM‐B decreased VEGF induced HMVEC chemotaxis (n=8), and inhibited VEGF induced HMVEC tube formation on Matrigel (n=9). Using Western blotting, we also found that sJAM‐B exerts its effect by stimulating the phosphorylation of Erk in a time dependent fashion (n=2). Our results indicate that sJAM‐B inhibits sJAM‐C and VEGF induced angiogenesis.
Emerging role of epigenetic in pulmonary arterial hypertension right ventricular failure
Authors: Potus Francois , Ruffenach Gregoire , Provencher Steeve , Bonnet Sébastien
Published: June 2021

INTRODUCTION

Pulmonary arterial hypertension (PAH) is associated with a global impairment of angiogenesis. Although the origin of the angiogenic defect remains unknown. since a few years epigenetic has become an important research area in many diseases including PAH. Role for miRNAs has been widely demonstrated; nonetheless a role for long noncoding RNA (LncRNA) remains unknown. In the present study we focused our role on the expression level of miR‐126 and MALAT‐1 both of which are enriched in endothelial cells and known to be implicated in angiogenesis regulation. We hypothesized that aberrantly express miR‐126 and MALAT‐1 contributes to angiogenesis impairment see in PAH.

RESULTS

Using qRT‐PCR we showed that miR126 was decreased in human PAH RV (p<0.05) compare to control while MALAT‐1 expression was upregulated p<0.05). These finding were associated in vitro with a significant reduction in EC proliferation (Ki67;p<0.05) and tube formation (p<0.05) measured in freshly isolated human RV endothelial cells from both PAH compare to EC from control RV. Both miR‐126 and MALAT‐1 are critical regulators of angiogenesis and are likely dependent of each other; in fact we showed that miR‐126 expression inversely correlates with MALAT‐1 expression in PAH RV (p<0.05).

CONCLUSION

We demonstrated, for the first time in PAH that the endothelial‐enriched noncoding RNAs miR‐126 and MALAT‐1 are aberrantly expressed in human RV from PAH patients contributing to the impairment of angiogenesis. The endothelial specific miR‐126 is known to regulate endothelial cells proliferation while MALAT‐1 up regulation has been associated with decreased endothelial cells migration and sprouting, suggesting that these two noncoding RNAs are implicated in angiogenesis defect.
3‐Methylcholanthrene/Aryl‐Hydrocarbon Receptor‐Mediated Inactivation of eNOS through a RhoA/Akt‐dependent mechanism
Authors: Chang Chih‐Cheng , Juan Shu‐Hui
Published: June 2021

Endothelial nitric oxide synthase (eNOS) in endothelium modulates vascular blood pressure, and Akt/PKB mediates eNOS activation. We previously identified that activation of the aryl‐hydrocarbon receptor (AhR)/RhoA by 3‐methylcholanthrene (3MC) decreased PI3K/Akt phosphorylation. We studied the effect of 3MC and underlying mechanism of AhR/RhoA‐mediated the decrease in eNOS activity. Our results suggested that 3MC reduced the phosphorylation of eNOS through AhR/RhoA‐mediated Akt inactivation and decreased the eNOS‐Hsp90 complex formation in mouse cerebral vascular endothelial cells (MCVECs). Akt inactivation by 3MC decreased eNOS phosphorylation through an AhR/RhoA‐dependent pathway, accompanied by reducing the activated form of eNOS dimer. Immunofluorescence assay showed that membrane‐associated eNOS was decreased after 3MC challenge. NO production was determined using Griess assay, showing 3MC decreased NO production. Additionally, AhR/RhoA activation altered the mRNA and protein levels of Hsp90, Caveolin‐1, Dynein and eNOS. Co‐IP assay showed that protein interactions among Akt, Hsp90, and eNOS were significantly alleviated, but conversely the enhanced interaction of caveolin‐1 and eNOS was observed. Interestingly, siAhR, DNRhoA, and simvastatin reversed the effects of 3MC in MCVECs. In vivo, we observed that 3MC elevated blood pressure in mice, which was prevented by pre‐treatment of RhoA inhibitor, simvastatin, or nitric oxide synthase activator, SNP, suggesting the involvement of RhoA and NO in this event. Collectively, our studies show effective therapeutic approaches to recover 3MC‐mediated hypertension by simvastatin and SNP intervention. (NSC 102‐2320‐B‐038‐030‐MY3 (2‐3)).