bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Miscellaneous Genes Upregulated in Hepatocellular Carcinoma in Patients with Alcoholic (ASH) and Non‐alcoholic Steatohepatitis (NASH)
Authors: Lu Jiajie , Nguyen Luan , Samadzadeh Sara , Masouminia Maryam , Mendoza Alejandro , Tillman Brittany , Morgan Timothy , French Barbara , French Samuel
Published: June 2021

Both non‐alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) can lead to cirrhosis and hepatocellular carcinoma. However, the rate of progression to cirrhosis and tumorigenesis in ASH is greater than that in NASH. We asked whether there are differences between the two conditions in the expression levels of proteins involved in the pathogenesis of hepatocellular carcinoma. The proteins tested were presented at the 2017 AASLD Liver Meeting as overexpressed in hepatocellular carcinoma: KLF4, SCL19A1, FANCG, HRH‐1, DNMT1, DNMT3B, TNFR2, DUSP4, EGFR, Integrin, HDACII, and PDE3A. The expression of these proteins was measured in liver biopsy sections from NASH, ASH and normal patients using immunohistochemical staining with fluorescent antibodies and then quantifying the fluorescence intensity morphometrically. In ASH patients, levels of all tested proteins except HRH‐1 were elevated compared to normal patients. In NASH patients, KLF4, SCL19A1, FANCG, DNMT1, DNMT3B, and HDACII levels were increased compared to normal controls while HRH‐1 and PDE3A levels were decreased. The relative expression of all proteins studied except DNMT‐1 was higher in ASH compared to NASH. In conclusion, proteins involved in hepatocellular cancer development are more highly expressed in ASH compared to NASH and normal liver, which corresponds with the higher rate of tumorigenesis in ASH patients compared to NASH patients.

Support or Funding Information

This study was funded by NIH/AAA grant # UO‐21898‐05

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Carbon dioxide challenge enhances the sensitivity of whole body plethysmography (WBP) measurements of lung growth and ozone‐induced changes in juvenile Long‐Evans rats
Authors: Dye Janice , Stewart Erica , Ledbetter Allen , Miller Colette
Published: June 2021

Background

Risk factors associated with development of childhood disorders such as asthma and obesity include prematurity and intrauterine growth restriction. We recently demonstrated that exposure of Long Evans rats in early pregnancy to the air pollutant, ozone, resulted in reduced near‐term fetal growth. We propose to utilize this model of early life environmental stress to examine developmental origins of asthma later in life. Therefore, in this pilot study, we sought to improve the sensitivity of WBP to monitor lung volume growth in rats during development.

Methods

Subset A male and female offspring from air‐ or 0.8 ppm ozone‐exposed dams (4h/day on gestational day 5 and 6) (n ≅ 6/group) were monitored weekly from postnatal day (PND) 13 to PND70 for changes in body weight and spontaneous ventilatory parameters [i.e., breathing frequency (f), tidal volume (TV), minute volume (MV), EF50 and Penh]. Data were first obtained using mouse and later rat WBP chambers with EMKA iox 2 software (SCIREQ, Montreal, Canada). To enhance tidal breathing, Subset B male offspring (n = 7; ≥ PND50) were used to optimize a CO2 challenge protocol (5–7.5% CO2) for up to 10 minutes under control conditions, and then later (PND81) to assess whether the CO2 challenge would also be tolerated acutely following exposure to ozone (0.4 or 0.8 ppm ×4h).

Results

Subset A offspring underwent rapid growth during the 8‐week study period with nearly 15‐fold increases in body weight in males and 10‐fold increases in females. Corresponding increases in TV (~0.2 to > 2.0 mL) were observed. In Subset B males, a 6% CO2 challenge for 6 minutes appeared optimal. Likewise, in Subset A rats at PND67, this CO2 challenge resulted in more uniform breathing, less movement‐related artifacts, and increased ventilatory effort (TV increased ~50%); although females were somewhat less responsive. Lastly, use of the 6% CO2 challenge immediately following ozone exposure of Subset B males more readily distinguished the resultant breathing pattern alterations and lung volume deficits occurring.

Conclusions

Although the CO2‐enhanced lung volume measurements obtained herein are not equivalent to maximal, expiratory maneuvers (i.e., FEV1) as can be achieved in humans, this protocol was well tolerated and may provide a more sensitive, yet non‐invasive method of documenting differential lung growth trajectories in offspring— with or without prior in utero ozone exposure — as they undergo additional exposures during weanling and peri‐adolescent life stages. (Abstract does not reflect USEPA policy).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Patient‐Centered Clinical Toxicology Elective in Collaboration with the Illinois Poison Center
Authors: El‐Alfy Abir
Published: June 2021

Most didactic pharmacy school curricula hardly devote any part of the coursework to toxicology. However, appendix 1 of the 2016 Accreditation Standards lists toxicology topics to be covered in the didactic coursework. These topics include mechanisms, prevention, and treatment of toxicity of drugs and poisons, especially bioterrorism ones. A few hours in didactic curriculum usually satisfy this accreditation requirement. However, since pharmacists play an important role as first health responders, it was essential to develop a clinical toxicology elective course in our program. A 2‐hour course was established in collaboration with clinical pharmacists at the Illinois Poison Center, and offered for third year student pharmacists, during the Spring semester. The course was offered for four years, with average enrollment of 24 students/year. The course topics were tailored yearly according to the American Association of Poison Control Centers' Annual Report (AAPCC). Different active learning techniques were used over the four years. These included case presentations, small group case discussions, in class, and flipped classroom activities. The average student performance in the course for academic years 2011–2014 was 86.77 ± 0.89%. All cases discussed in the course were either provided by the Illinois Poison Center or retrieved from literature case reports. Students analyzed the cases, provided SOAP note reports, and presented them orally. Case presentation grade constituted 25% of the overall grade. The average student performance in the case analysis and presentation was 91.90 ± 0.90%. In conclusion, the toxicology elective developed in collaboration with the poison control center provided a clinically relevant, interactive experience to our students.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histochemical & Behavioral Analysis of the 3xTg‐AD Mouse Model of Alzheimer's Disease
Authors: Mitrano Darlene A. , Schendzielos Rachel , Croushore Emma , Genovese Benjamin , Quintanilla Ricardo , Dymond Ethan , Pearce Patrick , Houle Samuel , Halleck‐Pinkleton Hanna , Grau Harold , Webb Lisa Smith
Published: June 2021

Alzheimer's disease (AD) is an incurable neurodegenerative disease that affects about 11% of Americans age 65 and over and has very few FDA‐approved treatments. As the primary areas affected within the brain are the hippocampus and the cortex, the main reported symptoms of this disease are memory loss and confusion. However, the disease can also affect olfactory function, a symptom that develops prior to changes in cognitive function. Due to the lack of effective treatments, developing an animal model that most closely resembles human AD is crucial. The goal of this study was to further validate a mouse model of AD, the 3xTg‐AD mouse which expresses both the amyloid plaques and neurofibrillary tangles in the brain, to determine if the mice express olfactory deficits seen in humans. To do this, we used a buried food test in which mice were food deprived for 24 hours and then placed in a rat cage with a piece of rodent chow buried beneath the bedding. We timed, up to 15 minutes, how long it took the mice to uncover the buried food as a way to measure olfactory detection. The 3xTg‐AD mice were compared to their background and parental strains (C57Bl/6, 129Sv/E, and a hybrid of the C57Bl/6 and 129Sv/E). Preliminary results indicate that over the span of 52 weeks, the 3xTg‐AD mice take significantly longer than the other strains to find the buried food. In order to correlate the behavioral observations with anatomical changes, the Congo Red staining technique was employed to identify whether or not amyloid plaques develop in the olfactory bulbs of the mice. Thus far we have observed plaque accumulation in the glomerular layer of the olfactory bulbs of the 3xTg‐AD mice. Overall, the results of this study increase the validity of the 3xTg‐AD mouse as a model of human AD and leads to new areas of study to determine where else in the brain olfactory function is affected by AD.

Support or Funding Information

This work was supported in part by Award No. 17‐2 from the Commonwealth of Virginia's Alzheimer's and Related Diseases Research Award Fund, administered by the Virginia Center on Aging, School of Allied Health Professions, Virginia Commonwealth University (to D.A. Mitrano & L.S. Webb)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Angiotensinergic neurotransmission in the medial amygdaloid nucleus modulates the cardiovascular responses to emotional stress in rats.
Authors: Crestani CARLOS C. , Costa‐Ferreira Willian , Gomes‐de‐Souza Lucas
Published: June 2021

The physiological and behavioral responses observed during aversive threats are coodinated by overlapping circuits in the central nervous system. The medial amygdaloid nucleus (MeA) is a limbic structures implicated in the control of cardiovascular responses to stress. However, the local neurochemical mechanisms involved in such control has not been fully elucidated. In this sense, angiotensin II and their receptors were identified within the MeA. Nevertheless, a possible involvement of this neurochemical mechanism in MeA control of stress‐evoked cardiovascular responses has never been evaluated. Thus, the aim of this study was to investigate the involvement of AT1 receptor within the MeA in control of cardiovascular responses evoked by an acute session of restraint stress in rats. For this, male Wistar rats (240g–260g) had cannula‐guide bilaterally implanted within the MeA. A catheter was also implanted in the femoral artery for mean arterial pressure (MAP) and heart rate (HR) recording. The restraint stress was performed by placing the animals in a plastic cylindrical tube for 60 minutes. Independent group of animals received bilateral microinjections into the MeA of the selective AT1 receptor antagonist losartan (1nmol/100nL), angiotensin II (0.05nmol/100nL) or vehicle (saline, 100nL) 10 min before the onset of the restraint stress session. We observed that bilateral microinjection of losartan into the MeA enhanced the tachycardia evoked by restraint stress (F(1,11)=7, P<0.02), but without affecting the MAP increase (F(1,11)=1, P>0.05). Similarly, bilateral treatment of the Mea with angiotensin II enhanced restraint‐evoked HR increase (F(1,12)=6.5, P<0.02) without affecting the MAP response (F(1,12)=0.5, P>0.05). Taken together, these results provide evidence of an inhibitory role of the AT1 receptor within the MeA in cardiac responses to emotional stress. Besides, results indicate that angiotensin II acting via mechanisms other than the AT1 receptor plays a facilitatory influence on HR response.

Support or Funding Information

FAPESP, CNPq and PADC‐FCF/UNESP.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The α7 nicotinic receptor positive allosteric modulator, PNU‐120596, reverses lipopolysaccharide‐induced depressive‐like behavior in mice
Authors: Alzarea Sami , Rahman Shafiqur
Published: June 2021

Previous evidence indicates that neuroinflammation plays a critical role in the pathophysiology of major depressive disorder (MDD) involving microglial α7 nicotinic acetylcholine receptors (nAChRs). The objective of the present study was to determine the effects of PNU‐120596, an α7 nAChR positive allosteric modulator (PAM), on depressive‐like behavior and expression of ionized calcium binding adaptor molecule 1 (Iba‐1), microglial marker, in mice following lipopolysaccharide (LPS) administration, an animal model for depressive‐like behavior. Forced swim test (FST) and tail suspension test (TST) were used to determine the effects of PNU‐120596 on depressive‐like behavior. We also examined the effects of PNU‐120596 on Iba‐1 expression by using Western blot analysis and immunofluorescence staining in the hippocampus and prefrontal cortex, brain regions implicated in MDD. Administration of LPS (1 mg/kg, i.p.) significantly increased immobility time during FST and TST. The PNU‐120596 (1 or 4 mg/kg, i.p.) dose‐dependently decreased the LPS‐induced immobility time during FST and TST. Similarly, the PNU‐120596 (4 mg/kg, i.p.) significantly decreased the LPS‐induced increased Iba‐1 expression in the hippocampus and prefrontal cortex. In conclusion, these results suggest that PNU‐120596 reduces LPS‐induced depressive‐like behavior likely by targeting microglial α7 nAChRs in the hippocampus and prefrontal cortex in mice. Therefore, α7 nAChR PAMs could be developed as potential therapeutic utility for the treatment of MDD in humans.

Support or Funding Information

Supported in part by grant from Saudi Arabian Cultural Mission, USA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AMPA receptor modulators selective for the accessory protein TARP‐g8
Authors: Maher Michael P. , Wu Nyantsz , Ravula Suchitra , Ameriks Michael K. , Savall Brad M. , Lord Brian , Seierstad Mark , Carruthers Nicholas I. , Lovenberg Timothy W.
Published: June 2021

Background

The α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors mediates the majority of fast synaptic transmission within the mammalian brain. The ubiquitous expression of the primary subunits of AMPA receptors (AMPARs), and the lack of pharmacological selectivity amongst them, preclude regional or neuronal subtype specificity. In vivo, AMPARs comprise a variety of accessory proteins. Of particular interest, TARP‐γ8 is highly expressed in the hippocampus, a region of the brain implicated in several psychiatric and neurological disorders.

Methods

We used high‐throughput screening to discover compounds that selectively modulate AMPARs containing TARP‐γ8. Subsequent medicinal chemistry efforts were used to improve potency and pharmacokinetics of the hits. Assays were developed to measure target occupancy and functional effects of the compounds in vivo.

Results

These compounds possess a novel mechanism‐of‐action consistent with a partial attenuation of the interaction between the TARP and the pore‐forming subunits of the channel. Lead molecules with oral bioavailability and high brain penetration allowed demonstration of a strong relationship between pharmacokinetics and pharmacodynamics. The compounds show anticonvulsant and anxiolytic profiles in rodent. Molecules in this class provide large safety margins in preclinical species relative to non‐specific AMPAR antagonists due to the improved regional specificity of TARP‐γ8 modulators.

Conclusions

AMPAR modulators selective for TARP‐γ8 have the potential to be novel treatments for anxiety/depression, bipolar disorder, temporal lobe epilepsy, and/or prodromal schizophrenia. This project also represents proof‐of‐concept for pharmacological targeting of accessory proteins and small‐molecule modulation of protein‐protein interactions.

Support or Funding Information

Authors are employees of Janssen R & D, LLC

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ligand Binding to the β2‐Adrenergic Receptor is Dependent Upon its Oxidation State
Authors: Rambacher Kalyn M. , Moniri Nader H.
Published: June 2021

Inhaled β2‐adrenergic receptor (β2AR) agonists are the gold standard treatment of an acute asthma attack and function by eliciting rapid bronchial dilation. Asthmatic tissue is characterized by increased expression of NADPH Oxidase (NOX) and subsequent increased levels of reactive oxygen species (ROS) compared to healthy tissue. We and others have previously showed that agonism of β2AR with ‐isoproterenol (ISO) leads to activation of NOX, and generation of intracellular ROS. We have also previously shown that these ROS are capable of oxidizing cysteine residues on the β2AR itself, a process known as cysteine‐S‐sulfenation. Emerging evidence has established cysteine‐S‐sulfenation as a functionally significant post translational protein modification, which thus far has not been evaluated for its effects on β2AR ligand binding. Utilizing isolated plasma membrane and whole cell radioligand binding, the current study evaluates oxidation state‐dependent alterations to ligand binding to the β2AR in HEK293 cells over‐expressing β2AR and in immortalized human lung epithelial cells. Specific ligand binding is significantly increased in the oxidized receptor state in both cell lines and in both membrane and whole cell assays. This effect is reversed by receptor labeling with the sulfenic acid selective small molecule dimedone, indicating increased ligand binding is specific to the presence of cysteine sulfenic acids on the β2AR. Furthermore, isoproterenol can fully displace radioligand binding in all redox states to the same extent in a monophasic manner, indicating redox state either directly or indirectly modifies the β2AR's orthosteric binding pocket. Given the oxidative environment native to asthmatic tissue, the findings presented here have implications for asthma treatment with β2AR agonists, and present a need to better understand how β2AR may interact differently with ligands in asthmatic tissue compared to healthy models.

Support or Funding Information

This work was funded by the NIH/NHLBI grant HL138603 to NHM.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure Assisted Design of Small Molecule KCa Channel Modulators
Authors: Wulff Heike , Nguyen Hai M. , Brown Brandon M. , Shim Heesung , Yarov‐Yarovoy Vladimir
Published: June 2021

Pharmacological modulation of ion channels offers tremendous opportunities for the design of new drugs for neurological and cardiovascular diseases. However, in contrast to other therapeutic targets such as protein kinases, ion channels have not been popular with medicinal chemists in the pharmaceutical industry. One reason is the absence of co‐crystals of medically relevant channels with bound drug molecules, a prerequisite for structure based drug design. Using the Rosetta molecular modeling suite we generated homology models of the pore of the Ca2+‐activated K+ channel KCa3.1 and tested the models by first confirming previously mapped binding sites and visualizing the mechanism of TRAM‐34, senicapoc and NS6180 inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K+ before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4‐phenyl‐pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4‐phenyl‐pyran in the pore lumen, which could both be confirmed by site‐directed mutagenesis and electrophysiology. We further used Rosetta modeling to explain the KCa3.1 selectivity of the positive allosteric gating modulator SKA‐121, a compound that binds at the interface of the calmodulin N‐lobe and the calmodulin binding domain of these voltage‐independent Ca2+‐activated channels, where it “facilitates” mechanical opening (= increase open channel probability) at a given Ca2+ concentration. Rosetta Ligand provides a plausible explanation for why KCa channel activators in general are 5–10‐fold more potent in activating KCa3.1 than KCa2 channels. The presence of R362 creates an extensive “background” hydrogen‐bond network in the KCa3.1 interface pocket that stabilizes the main contacts NH2‐substituted KCa activators make with M51 and E54 in calmodulin. The three KCa2 channels have shorter N or S residues in the corresponding position and therefore cannot form this hydrogen‐bond network.

Overall these examples illustrate that ion channels are highly accessible to small molecule drugs and we suggest that both the KCa3.1 pore and KCa3.1‐CaM‐BD/CaM model could be used for structure assisted drug design.

Support or Funding Information

This work was supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), Grant Number U54NS079202 and R21NS101876 (to H.W.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Soluble Epoxide Hydrolase/Phosphodiesterase Dual Inhibitor is Efficacious Against Inflammatory Pain
Authors: Wagner K. M. , Bloecher Rene , Reddy Gopireddy R. , Morisseau Christophe , Hammock Bruce D.
Published: June 2021

The soluble epoxide hydrolase (sEH) is the principal regulator of epoxy fatty‐acids (EpFA) transforming them in to the less active diols. Inhibitors of sEH block this activity and maintain levels of EpFA in vivo which has demonstrated potent analgesia in several preclinical models of acute and chronic pain. However, sEH inhibitors have no observable action in the absence of a pain state [1]. It was therefore hypothesized that an additional factor was present in the painful state that was required for the antinociceptive action of the EpFA [2]. Investigation began with cyclic adenosine monophosphate (cAMP) and experiments demonstrated this was active but also that increasing cAMP with phosphodiesterase inhibitors increased EpFA as well. Phosphodiesterase enzymes hydrolyze the cyclic nucleotide into adenosine monophosphate altering its secondary messenger capacity. This led to the design and synthesis of several dual inhibitors of sEH and phosphodiesterase.

Multi‐target ligands are designed to improve safety and efficacy in treatment of diseases. The use of drug combinations cannot be assumed to be safe given drug‐drug interactions. Additionally, the prediction of pharmacodynamic and pharmacokinetic relationships is less complicated with a single entity. The multi‐target ligands are selected to improve therapeutic efficacy by hitting multiple targets in physiological disorders. Thus, increasing the EpFA by halting their degradation and at the same time potentiating their activity with an increase in cAMP was the goal with synthesis of the dual inhibitor. We present the biochemical characterization as well as the in vivo efficacy in an inflammatory pain model for the selected dual inhibitor of sEH and PDE enzymes. The IC50 of RBH61 for sEH determined on the sEH human recombinant enzyme is 2.1 ± 0.5 nM. This is paired with a IC50 in the PDE assay of 8.1 ± 0.05 nM in human embryonic kidney (HEK293) cells. Evaluation of the dual inhibitor in a model of inflammatory pain in rats demonstrates its antinociceptive efficacy.

Support or Funding Information

This work was supported, in part, by National Institutes of Health grants: NIEHS R01 ES002710 and NIEHS/Superfund Research Program P42 ES004699, NINDS U54 NS079202 and Grants NIEHS T32ES007059, NIH 5T32DC008072‐05 and 4T32HL086350‐09.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of a Novel Inhibitor of Soluble Epoxide Hydrolase and Role in Ocular Neovascularization
Authors: Park Bomina , Pasha Sheik Pran Babu Sardar , Si Yubing , Meroueh Samy O. , Seo Seung‐Yong , Corson Timothy W.
Published: June 2021

Objective

Ocular neovascularization causes blindness in diseases such as proliferative diabetic retinopathy (PDR) and wet age‐related macular degeneration (AMD). Today, the effort to treat wet AMD is hampered by patients who are resistant and refractory to the current standard of care, anti‐vascular endothelial growth factor (VEGF) therapies. Therefore, identification of novel therapeutic targets and inhibitors is desirable to address the unmet needs in antiangiogenic treatment. Previously, we have identified soluble epoxide hydrolase (sEH) as a binding target of an antiangiogenic small molecule, SH‐11037. Here, we aimed to further investigate sEH expression in the eye and the inhibition of its activity by SH‐11037.

Methods

The expression and localization of sEH in retinal sections of laser‐induced choroidal neovascularization (L‐CNV) mice were evaluated by immunohistochemistry. The sEH enzymatic activity in ocular tissue homogenates of L‐CNV and control mice was assayed using trans‐stilbene oxide (t‐SO) as substrate. The sEH inhibition by SH‐11037 and mode of inhibition was assessed by recombinant sEH activity assay using a fluorogenic substrate, PHOME. Docking and molecular dynamics simulations were performed to predict the binding mode of SH‐11037 within the active site of sEH. In addition, intravitreal injections of sEH substrate 19,20‐epoxy docosapentaenoic acid (EDP) and product 19,20‐dihydroxy docosapentaenoic acids (DHDP) were given to L‐CNV mice, and effects were assessed by imaging and choroidal flatmount immunofluorescence.

Results

sEH was upregulated in the rod photoreceptors in L‐CNV mice compared to control mice and it did not co‐localize with other retinal cell type markers. Correspondingly, sEH activity was increased in L‐CNV eyes and was normalized by SH‐11037 or a known sEH inhibitor. In a recombinant enzyme assay, SH‐11037 inhibited sEH activity in a dose‐dependent manner, with IC50 = 0.15 μM. Enzyme kinetics analysis demonstrated that increasing concentrations of SH‐11037 decreased Vmax and increased KM, revealing SH‐11037 as a mixed‐type inhibitor of sEH, with Ki = 1.73 ± 0.45 μM. Docking of SH‐11037 to sEH showed an energetically favorable binding mode with the ligand spanning the active site of the enzyme and interacting with key catalytic residues. Intravitreal 19,20‐EDP reduced CNV lesion volume compared to vehicle and 19,20‐DHDP, confirming antiangiogenic properties of this sEH substrate.

Conclusion

Our studies confirm the relevance of sEH for ocular neovascularization and reveal the mechanism for SH‐11037's inhibition of sEH. Further optimization of SH‐11037's structure could yield more potent, novel sEH inhibitors.

Support or Funding Information

NIH/NEI R01EY025641, Retina Research Foundation, Research to Prevent Blindness, Inc.
Modulation of Arachidonic Acid Metabolism in the Rat Kidney by Quercetin: Implications for Regulation of Blood Pressure
Authors: Elbarbry Fawzy , Harrelson John , Espiritu Michael , Pham Therese , Ibrahim Khaled
Published: June 2021

Background

We investigated the effects of quercetin (QR), a plant polyphenol from the flavonoid group, on Arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously hypertensive rats (SHR) as our animal model system.

Methods

Rats were treated for 7 weeks with QR (10, 30, or 60 mg/L) added to their drinking water; drinking water alone was used as our control. Mean arterial pressure (MAP) was measured at 7‐day intervals throughout the study. At the end of the treatment, rats were euthanized, followed by preparation of kidney microsomes and cytosols to measure the activity (using LC/MS/MS) and expression (using immunoblotting) of enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the formation of 20‐hydroxyeicosatetraenoic acid, and the soluble epoxide hydrolase (sEH), which is responsible for the degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids.

Results

We found that treatment with QR lead to significant reductions in both the expression and activity of renal CYP4A isozyme. Only the high dose QR resulted in a significant reduction in the expression, but not the activity, of renal sEH. Consistent with these data, we have found that treatment with QR resisted the progressive rise in MAP in the developing SHR in a dose‐dependent manner.

Conclusion

This is the first demonstration that QR modulates the metabolism of AA by both P450 enzymes and sEH in SHR rats. This may represent a novel mechanism by which QR protects SHR rats against the progressive rise in blood pressure.

Support or Funding Information

Pacific University Research Incentive Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alterations in the Eicosanoid Profile and Mitochondrial Injury in Human Ventricular Tissue Following Myocardial Infarction
Authors: Jamieson Lockhart , Shah Saumya , Edin Matthew , Zeldin Darryl , Oudit Gavin Y. , Seubert John M.
Published: June 2021

Study objective

Myocardial infarction (MI) accounts for a significant proportion of death and disability in the ageing population. n‐6 PUFAs linoleic acid and arachidonic acid have come under investigation for their role in mediating a cardiac response following ischemic injury. CYP450 metabolism of these fatty acids results in formation of numerous metabolites, called eicosanoids, which can be further metabolised by the enzyme soluble epoxide hydrolase (sEH). These lipid mediators exhibit a wide range of cellular effects including alterations to mitochondria structure and function which may impact cardiac performance. This study investigated alterations to CYP‐derived eicosanoids and mitochondria in heart tissue obtained from individuals who experienced a previous MI.

Methods

Samples were obtained from male and female individuals included in the Human Explanted Heart Program and correlated to non‐failing control hearts (NFC) collected from unused transplant donors through the Human Organ Procurement and Exchange Program at the University of Alberta. Ventricular tissues were harvested from patients who had previously experienced a LAD infarct (≥2yrs), as determined by echocardiography, ECG and coronary angiograms. Protein expression was determined by immunoblotting techniques. Mitochondrial enzymatic activities were assessed by spectrophotometric methods while mitochondrial ultrastructure and cristae density were assessed by electron microscopy. A metabolic profile in ventricular tissue was obtained by LCMS.

Results

Marked differences in both LA and AA metabolic profiles were revealed in post‐MI tissues. There were significant increases in cardiotoxic metabolites correlating with decreased cardiac function and injury. Interestingly, there was a significant increase in both sEH activity and expression in post‐MI tissues compared to NFC. Expression of mitochondrial proteins remained unchanged, however enzymatic function declined in post‐MI tissues compared to NFC correlating with marked disruption in mitochondrial ultrastructure.

Conclusions

These data provide the first evidence demonstrating a marked shift in eicosanoid metabolism in post‐MI hearts that correlates with mitochondrial structural disruption and overall decreased mitochondrial function.

Support or Funding Information

Support for this project was provided by the Canadian Institutes of Health Research.

KL Jamieson is supported by funding from Alberta Innovates‐Health Solutions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EETs exacerbate chronic hypoxia‐induced pulmonary hypertension
Authors: Kandhi Sharath , Alruwaili Norah , Froogh Ghezal , Yang Yang‐Ming , Deng Wensheng , Wolin Michael S. , Huang An , Sun Dong
Published: June 2021

We have demonstrated that epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acids that are degraded by soluble epoxide hydrolase (sEH), initiate increases in right ventricular systolic pressure (RVSP) by perhaps, increasing pulmonary vascular resistance. Also, EETs enhance acute hypoxic pulmonary vasoconstriction (HPV) by shifting the balance between constrictor and dilator prostaglandins towards constrictor prostanoids. Prolonged HPV promotes hypoxia‐induced pulmonary hypertension (HPH), we therefore, tested the hypothesis that deletion of sEH gene (sEH‐KO) promotes EET‐mediated potentiation of HPH.

Methods

Male sEH‐KO (12‐weel old) and age matched C57BL/6J (WT) mice were exposed to 10% O2 for 21 days. Cardiac hemodynamics were assessed by echocardiography and right heart catheterization. Right ventricular hypertrophy (RVH) was expressed as the weight ratio of right ventricle (RV) to left ventricle (LV) with septum (RV/LV+S). sEH expression in pulmonary tissue was assessed by western blot. Pulmonary EET levels were measured by LC‐MS/MS analysis.

Results

Pulmonary doppler flow echocardiography showed that hypoxia elicited decreases in pulmonary arterial acceleration time (PAAT), ejection time (ET), and PAAT/ET ratio. The reduction is significantly greater in sEH‐KO mice compared to that of WT mice. Baseline RVSP was comparable between WT and sEH‐KO mice. Hypoxia exposure significantly increased RVSP in both strains of mice, the increment however, was significantly greater, associated with greater RVH in sEH‐KO mice compared to their WT controls. This indicates that sEH‐KO mice developed more severe HPH. Although chronic hypoxia also downregulated pulmonary sEH expression in WT mice, sEH‐KO mice exhibited highest level of EETs that correlated to their greater increases in RVSP and more severe HPH. In conclusion, the deficiency of sEH promotes EET‐mediated exacerbation of HPH.

Support or Funding Information

(This work was supported by HL070653, HL115124 and HL129797).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Soluble epoxide hydrolase contributes to the endothelial dysfunction of peripheral conduit arteries in type 2 diabetic patients
Authors: Duflot Thomas , Remy‐Jouet Isabelle , Morisseau Christophe , Wils Julien , Li Dongyang , Prevost Gaetan , Joannides Robinson , Bellien Jeremy
Published: June 2021

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes the vasodilator and anti‐inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), contributes to conduit artery endothelial dysfunction in type 2 diabetic patients.

We measured radial artery endothelium‐dependent flow‐mediated dilatation in 29 patients with well‐controlled type 2 diabetes (HbA1c=6.7±0.6%) and 37 control subjects in response to hand skin heating. We quantified the local plasma concentrations of EETs, DHETs, the NO metabolite nitrite and reactive oxygen species (ROS) at baseline and during heating. The sEH protein expression and activity were measured in peripheral blood mononuclear cells (PBMCs) isolated from patients and controls.

Flow‐mediated dilatation was reduced in diabetic patients compared to controls, without change in flow variation and endothelium‐independent dilatation to glyceryl trinitrate. Diabetic patients displayed an elevation in ROS levels and a complete loss of NO release and in EETs production, assessed by the sum of total EETs and DHETs, during the flow stimulation. The conversion of EETs to DHETs (DHET‐to‐EET ratio) was enhanced at baseline and further increased during heating in diabetic patients compared to controls. Moreover, sEH expression and activity were increased in diabetic PBMCs.

This study demonstrates that an increased degradation of EETs by sEH contributes with altered NO bioavailability to conduit artery endothelial dysfunction in type 2 diabetic patients. Inhibitors of sEH may be useful to improve endothelial function and thus cardiovascular prognosis in these patients.

Support or Funding Information

This study was funded by a grant from the Fondation de France (2011‐20459) and supported by National Institute of Environmental Health Sciences grant R01‐ES002710.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of the Dipeptidyl Peptidase‐4 Inhibitor Sitagliptin on Gentamicin Nephrotoxicity in Mice
Authors: Ali Badreldin H. , Suleimani Yousuf M. , Abdelrahman Aly M. , Karaca Turan , Nemmar Abderrahim
Published: June 2021

This study aimed at investigating the possible ameliorative effects of sitagliptin in mice with gentamicin (GEN) nephrotoxicity. Sitagliptin was given to the animals at an oral dose of 10 mg kg−1 per day for 10 days, and in some of these mice, GEN was injected intraperitoneally at a dose of 100 mg kg−1 per day during the last seven days of the treatment. Nephrotoxicity was evaluated histopathologically by light microscopy and biochemically by measuring several indices in plasma, urine and renal cortex homogenates. GEN treatment induced nephrotoxicity as evidenced by significantly (P<0.0001) increasing the plasma concentrations of urea, creatinine, circulatory cytokines, cystatin C, sclerostin, and TNFα. Treatment with GEN also significantly elevated urinary N‐acetyl‐β‐D glucosaminidase (NAG) concentration (P <0.0001). Moreover, GEN caused significant increase in oxidative stress in the kidneys (P<0.0001). Histopathological examination revealed massive tubular injury, necrosis, infiltration of inflammatory cells and intraluminal hyaline casts in mice treated with GEN. Sitagliptin alone did not significantly affect any of the indices measured. However, concomitant treatment with sitagliptin and GEN significantly mitigated most of the nephrotoxic actions of GEN. Pending further studies, sitagliptin may potentially be useful as a nephroprotectant agent.

Support or Funding Information

This work was financially supported by a grant from SQU, Oman (IG/MED/PHAR/17/1)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insights into the Structural Determinants of Substrate Binding to Human Cytochromes P450 2C9 and 2C18
Authors: Shah Manish B.
Published: June 2021

Xenobiotic metabolizing cytochrome P450 (CYP) enzymes constitute the major enzyme family in drug metabolism with increasing importance in pharmacogenetics, and more recently to pharmacogenomics. The highly polymorphic CYP2C9 metabolizes over 15% of clinical drugs that include ibuprofen, diclofenac, warfarin, losartan, tolbutamide, and glimepiride. Whereas CYP2C18 represents the least characterized enzyme with little information regarding substrates, the active site and the overall structure, despite the high sequence identity (more than 80%) with other human CYP2C subfamily of enzymes. Sequence alignment of CYP2C9 and 2C18 revealed interesting differences in amino acid substitutions, which allowed analysis of the active site of two enzymes of the same subfamily with distinct information regarding substrate specificity. A homology model of CYP2C18 created using the most recent crystal structure of CYP2C9‐losartan complex revealed key residue substitutions that significantly reduced the volume of the active site. In particular, the larger side chain substitutions L113, F201, F237 and N365 in CYP2C18 protrude the active site and play an important role in ligand interaction. Molecular docking studies using a diverse range of CYP2C9 substrates demonstrated remarkably altered orientation in the CYP2C18 active site indicative of differences in substrate preference compared to CYP2C9. The results suggest the active site architecture of CYP2C18 may not allow binding of larger or bulkier substrates as seen with other human CYP2C enzymes. Overall, the analysis of amino acid differences in the active site, the access channel and the recently identified potential substrate recognition site at the periphery provide important insights into the role of distinct side chain substitutions among related CYP enzymes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cytochromes P450 Involved in Metabolism of Flavonoid Aglycones
Authors: Bojic Mirza , Benkovic Goran , Males Zeljan , Zulj Rajka Truban , Tomic Sinisa
Published: June 2021

Flavonoids are secondary plant metabolites to which we are exposed through everyday diet (vegetables, fruits, dietary supplements, herbal drugs). They represent a wide class of compounds that have extensively been studied due to beneficial biological effects. As we have evolutionary been exposed to these compounds, human body has developed mechanisms how to excrete these compounds (metabolism, transporters). Objective of this study was to determine cytochromes P450 involved in metabolism of flavonoid aglycones and characterize generated metabolites. Metabolism of 30 flavonoids was assessed using human liver microsomes and recombinant liver cytochrome P450 enzymes. HPLC method coupled with UV detector and MS TOF was used for detection and quantification. Flavonoids could be separated based on retention time even though there are subtle differences in flavonoid structures. Structures of metabolites were elucidated using accurate mass data, retention time and confirmed using flavonoid standards. Flavonoid aglycones are susceptible to hydroxylation and demethylation reactions. The most significant enzyme involved in metabolism of analyzed flavonoids was cytochrome P450 1A2 which is in accordance with previously reported data. Other enzymes involved in metabolism of flavonoid aglycones were cytochromes P450 2D6, 2C19, and 3A4. As data on pharmacokinetics of flavonoids is limited to few well studied flavonoids, understanding metabolism of flavonoids will improve efforts to obtain flavonoid candidates less susceptible to metabolic elimination.

Support or Funding Information

This work was supported by the Croatian Science Foundation under the project UIP‐2014‐09‐5704 (M.B.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Chick Chorioallantoic Membrane (CAM) as a Multi‐Purpose Preclinical Model in Oncology
Authors: Hafner Susanne , Zuo Zhi , Rasche Volker , Syrovets Tatiana , Simmet Thomas
Published: June 2021

The chorioallantoic membrane (CAM) of fertilized chick eggs is a preclinical in vivo model and an alternative or pretest for animal studies in rodents. Although the CAM xenotransplantation model is a reproducible, reliable and efficient model conforming to the 3R principle to reduce mammalian animal experiments, and the United States Food and Drug Administration recommended the CAM for in vivo studies on angiogenesis, the method is not very common so far. Undoubtedly, the CAM assay has its limitations: The experimental window of about 10 days is a limiting factor hampering the establishment of the model in oncology. Here we show the implementation of the CAM model in oncological research on modern diagnostics and therapeutics. Tumor cells were xenotransplanted onto the chorioallantoic membrane, which serves as nutritious natural substrate for their growth and allows the three‐dimensional formation of solid tumors in an in vivo microenvironment. We successfully transplanted cancer cells stably expressing firefly luciferase, e.g. breast and lung cancer cells, and established methods for direct in ovo monitoring after the topical or intravenous administration of diagnostics or therapeutics. Upon administration of doxorubicin, a commonly used chemotherapeutic, the growth of cancer xenografts on the CAM was monitored by bioluminescence imaging using an IVIS in vivo Imaging System after the application of D‐luciferin for up to 3 days after treatment. The obtained results were quantified and validated by immunohistochemical analysis of proliferation and apoptosis (Ki‐67, Tdt) upon tumor tissue extraction. After labelling of compounds with gadolinium (Gd) as a contrast agent, high‐resolution magnetic resonance (MR) imaging at several time‐points enabled the evaluation of the compound's bio‐distribution and accumulation in tumor tissue. To confirm the MR findings, organs were extracted and analyzed for the Gd contents by inductively coupled plasma optical emission spectrometry (ICP‐OES).

These methods facilitate the straightforward and non‐invasive assessment of a compound's therapeutic efficacy and biodistribution and therefore possess implications for the development of new pharmacological substances.

The studies were conducted in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education.

Support or Funding Information

This work was supported by the Volkswagen Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.