bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of small molecule ligands targeting GPR83, a G‐protein coupled receptor activated by the abundant neuropeptide PEN
Authors: Lueptow Lindsay M. , Gomes Ivone , Gupta Achla , Sanchez Roberto , Devi Lakshmi
Published: June 2021

The G‐protein coupled receptor GPR83 is expressed in numerous brain regions, including striatum and hypothalamus, as well as in the immune system, including the thymus and spleen, of mice. Previously, GPR83 was classified as an orphan GPCR with no known endogenous ligand; we identified the neuropeptide PEN, derived from the precursor ProSAAS, as a selective, high‐affinity endogenous ligand for GPR83. Previous studies using ProSAAS or GPR83 knockout models have shown both the neuropeptide and receptor to be important in modulation of stress and anxiety, as well as feeding and body weight regulation. Additionally, the expression of GPR83 on immune cells indicates a potential role in immune function. Therefore, development of small molecule ligands for GPR83 is necessary in order to further our understanding of the receptor in these biological processes. In order to identify potential agonists and antagonists, a homology model for GPR83, based on the crystal structure of phylogenetically related receptors, was generated and used to screen in silico with the eMolecules library, which consists of 7 million compounds. From these, the top fifty predicted hits were screened in a cell based, high‐throughput calcium‐release assay. The compounds that gave a large signal to noise ratio only in cells expressing GPR83 were further characterized using GTPγS, adenylyl cyclase, phospholipase C, and receptor trafficking assays. This led to the identification of CPD1 and CPD27as selective agonists. Those compounds that did not give an agonistic response were re‐screened in an antagonist mode (i.e., in the presence of PEN, the endogenous agonist). The compounds that blocked PEN‐mediated calcium increase were further tested in GTPγS, adenylyl cyclase and receptor trafficking assays in the antagonist mode. From these assays, we identified CPD25 as a GPR83‐selective antagonist. Currently, we are characterizing these compounds using the native receptors in the brain and immune cells, with tissues from mice lacking GPR83 as controls. Characterization of these small molecule compounds provides much needed tools for furthering our understanding of the precise role of GPR83 in these biological processes. Also, since GPR83 has been highly implicated in the regulation of stress, mood and reward‐related behaviors, as well as immune function, identification of GPR83 selective ligands opens the door to development of novel therapeutics as regulators of this neuropeptide‐receptor system.

Support or Funding Information

Funding: NS026880 and DA019521 to L.A.D and T32 grant DA007135 to L.M.L.

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‐activity relationships of a distinct family of selective agonists of α7‐nAChRs
Authors: Camacho‐Hernandez Gisela Andrea , Kaczanowska Katarzyna , Doan Lisa , Bindig Hanna , Stokes Clare , Papke Roger L. , Taylor Palmer
Published: June 2021

Nicotinic acetylcholine receptors (nAChRs) are lead members of a pentameric ligand‐gated ion channel family; they are excitatory receptors, activated by the endogenous neurotransmitter, acetylcholine. nAChRs are present in the peripheral and central nervous system, where they mediate and modulate neuromuscular, autonomic and CNS functions. Receptor‐selective ligands that cross the blood‐brain barriers may form useful therapies for disorders of aging, such as Alzheimers disease and disorders of development, such as schizophrenia. In previous work, we developed a small sub‐group of 4,6 di‐substituted 2‐aminopyrimidines that interact with the acetylcholine binding protein (AChBP) and activate a7‐nAChRs. These agents show Kd and EC50 values for both AChBP binding and a7‐nAChR activation between 10 and 70nM. Moreover, they are structurally distinct from classical nicotinic agonists that are quaternary amines or typically contain a strong base separated from a weaker base or hydrogen bonding acceptor, often on separate ring systems. Selectivity for the a7‐AChR and high affinity for AChBP was achieved by substituting a dipicolyl group at the 4 position of the pyrimidine. In contrast to classical nicotinic agonists, the nitrogens on the pyrimidine and pyridine rings are weakly basic. Moreover, X‐ray crystallographic studies show that these ligands bind to the classical agonist‐antagonist site, but with a distinctive orientation of the core pyrimidine ring and the associated picolyl moiety. We have expanded our structural landscape of these AChBP binding and a7‐selective ligands, based on our previous leads by combining the pyrimidine system with the di‐picolyl substitution at the 4‐position. To examine receptor interactions of this family of compounds on nAChRs and related pentameric ligand‐gated ion channels, we employ HEK cells transfected with cDNA's encoding one of three requisite receptor subtypes: α7‐nAChR, α4β2‐nAChR and 5HT3AR, along with a fluorescent reporter. To assess rapid activation of lead candidates we use an oocyte system, revealing an agonist behavior distinguishing them from silent agonists and ago‐PAM's. To examine the molecular determinants and ligand poses involved, we have crystallized the higher affinity complexes with AChBP. Accordingly, we have a structure‐guided approach to novel ligand design for nAChRs. In conclusion, we find: 1) a di‐picolyl moiety in the 4‐position is fundamental for the activation of the a7‐nAChR, 2) the 2‐amino group tolerates only certain structural changes controlled by steric and bonding constraints, 3) substitutions at the 6‐position affect both the potency and receptor subtype selectivity of the ligands.

Support or Funding Information

CONACYT Mexico graduate fellowship to GACH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PEG‐peptide Scavenger Receptor Inhibitors for Non‐viral Gene
Authors: Allen Rondine , Rice Kevin , Mathew Basil
Published: June 2021

The rapid capture and degradation of viral and non‐viral gene delivery nanoparticles by scavenger receptors (SR) on Kupffer cells and fenestrated endothelial cells in the liver results in a decreased half life of all nanoparticles in an in vivo system, as well as a lower percentage of the dose available for delivery to the target organ. Current attempts to achieve SR inhibition involves co‐administering high molecular weight, polyanionic inhibitors. Polyinosinic acid (Poly‐I), the most widely used and potent inhibitor, competes for SR binding and successfully inhibits the uptake and metabolism of viral gene delivery nanoparticles in the liver. However, Poly‐I also activates the immune system, resulting in toxicity in mice, making Poly‐I clinically unacceptable.

We have discovered peptide based SR inhibitors that block SR uptake of DNA nanoparticles and improves their metabolic half‐life by forming protein nanoparticles in the blood.

Radio‐iodinated PEG‐peptides were used to study the pharmacokinetics and biodistribution to understand the structural properties which influence transfection competency and potency. We hypothesized that the in vivo potency and activity is influenced by the stability of protein nanoparticles formed in the blood. Results show that peptides featuring both hydrophobic and positively charged residues are able to potently inhibit scavenger receptor uptake of non viral gene delivery systems.

Support or Funding Information

This work was supported by the Iowa Biosciences Academy and University of Iowa Pharmacological Sciences T32 training grant. The Iowa Biosciences Academy is supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R25GM058939, the University of Iowa (UI) Office of the Vice President for Research and the UI Chief Diversity Office, the Pharmacological Sciences Training grant T32 GM067795 and NIH grant R01 GM087653.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Mechanistic Study on the Inhibition of Nicotine Metabolism by Trans‐Cinnamaldehyde
Authors: Espiritu Michael , Black Stephen , Johnston Casey , Chan Jeannine , Harrelson John
Published: June 2021

Trans‐cinnamaldehyde, a natural product found in cinnamon oil, has previously shown to selectively inhibit the major nicotine metabolizing enzyme cytochrome P450 2A6 (CYP2A6) via metabolism based inactivation using assays involving coumarin as a reporter substrate. In this study we aimed to investigate the mechanism by which CYP2A6 is inactivated by trans‐cinnamaldehyde, with the ultimate goal of developing trans‐cinnamaldehyde as a smoking cessation agent. We have also determined the level of inhibition of nicotine metabolism when nicotine is incubated with trans‐cinnamaldehyde, human liver microsomes and cytosol. CYP2A6 activity and heme loss were monitored in the presence of trans‐cinnamaldehyde and NADPH in vitro using human liver microsomes (HLM) and reconstituted CYP2A6 respectively. The effect of reactive oxygen species (ROS) generation on CYP2A6 activity and heme loss was studied using the reactive oxygen scavengers: N‐acetylcysteine, glutathione, catalase and superoxide dismutase. In an attempt to determine if heme loss was observed due to heme destruction or adduct formation, predictive multiple reaction monitoring (MRM) was performed using an optimized liquid chromatography tandem mass spectrometry (LC‐MS/MS) protocol. In activity studies using HLM and coumarin all reactive oxygen scavengers protected against activity loss, with N‐acetylcysteine and catalase offering the most protection (20% respectively) which, using a two tailed t‐test was found to be statistically significant with 99% confidence. Heme monitoring using LC‐MS/MS resulted in a significant amount of heme loss (97%) with a standard deviation (SD) of 3% after 18 minute incubations with trans‐cinnamaldehyde and NADPH in comparison to enzyme only controls. Inclusion of glutathione resulted in approximately 46% (SD of 7%) protection of the heme. Incubations with an NADPH regenerating system yielded a 41% (SD of 9%) loss of heme in comparison to controls without trans‐cinnamaldehyde. Predictive MRM yielded no detectable alkylated or acylated heme adducts or their respective oxidized forms. Results indicated an average inhibition of nicotine metabolism of 74.68% in the presence of trans‐cinnamaldehyde. This study demonstrated for the first time that CYP2A6‐mediated nicotine metabolism is inhibited by trans‐cinnamaldehyde and that ROS generation and heme destruction are major contributors to this process.

Support or Funding Information

Research reported here was supported by the National Institute on Drug Abuse of the National Institutes of Health under award number R15DA042341. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DISTRIBUTION, METABOLISM AND ELIMINATION OF BIA 10‐2474 IN THE RAT
Authors: Loureiro Ana I. , Fernandes‐Lopes Carlos , Moser Paul , Soares‐da‐Silva Patricio
Published: June 2021

In January 2016 one person died and others suffered adverse neurological effects during a Phase I clinical trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10‐2474. We have evaluated the distribution, metabolism and excretion of BIA 10‐2474 in the rat.

BIA 10‐2474 distribution and excretion was evaluated in Wistar rats after oral administration (30 mg/kg) of [14C]‐BIA 10‐2474. The metabolic profiling of BIA 10‐2474 was evaluated, in the same species, after oral administration of 90 mg/kg [14C]‐BIA 10‐2474.

The mean observed Cmax of total radioactivity in plasma occurred at 1 h and it was detectable up to 72h post‐dosing with 30 mg/kg [14C]‐BIA 10‐2474. The calculated apparent terminal half‐life of total radioactivity was 45h. Five days after oral dosing of Wistar rats, 97.6% (94% to 103%) of the administered dose had been excreted, with 65.2% (58.7% to 73.8%) of the radiolabelled material excreted in urine and 23.7% (20.8% to 29.1%) excreted in faeces. The amount of BIA 10‐2474 excreted in exhaled air was less than 0.4 %. The majority of the faecal excretion occurred in the first 24h post‐dose, representing about 83.1% of the excretion via this route. Similarly, the majority of urinary radioactivity was excreted in the first 24 h post‐dose representing at least 93.0% of the excretion via this route. The quantitative Whole‐Body Autoradiography (QWBA) studies in rat following single oral administration of [14C]‐BIA 10‐2474 (30 mg/kg), indicate that radioactivity was rapidly absorbed and distributed throughout the body. The high tissue to blood ratio of total radioactivity observed in the kidney medulla (9.46), pituitary gland (6.32), liver (5.70), kidney cortex (4.83), spleen (4.02), thymus (3.94), epididymis (3.81) and skeletal muscle (3.63) at 24 h post‐dose suggests a slower elimination of BIA 10‐2474 related radioactivity from these tissues. Following oral administration of 90 mg/kg [14C]‐BIA 10‐2474, the hydroxylation of the cyclohexyl ring of BIA 10‐2474 (BIA 10‐3827 and BIA 10‐2639), the N‐oxide reduced form of BIA 10‐2474 (BIA 10‐2445) followed by the hydroxylation in the cyclohexyl ring (BIA 10‐3764 and/or BIA 10‐2631) were identified. In brain homogenate samples, BIA 10‐2474, and metabolites BIA 10‐3764 and/or BIA 10‐2631, and BIA 10‐2445 have been identified.

In conclusion, BIA 10‐2474 was found to be rapidly absorbed and primarily excreted via the urine in the rat. The hydroxylation of the cyclohexyl ring of BIA 10‐2474 and BIA 10‐2445 was identified as the main metabolic route in the rat.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Novel Small Molecule Kinase Antagonists of the Prolactin Receptor In Breast Cancer
Authors: Bakshi Chinmay
Published: June 2021

Breast cancer remains to be a prevalent malignancy, as an estimated 231,840 women were diagnosed in the US in 2015. Furthermore, cancer cells have developed chemoresistance, necessitating the pursuit of innovative targets. Prolactin (PRL) acting via the prolactin receptor (PRLR) is an anti‐apoptotic hormone in breast cancer. Extensive research indicates that PRLR activation in breast cancer is associated with enhanced tumor growth, invasiveness, metastasis, and resistance to chemotherapy.

To identify novel antagonists of the PRLR, a 340,000 small molecule library was scanned using high throughput screening and computational modeling techniques (in‐silico docking) to observe binding potential to the PRLR. Kd and IC50 values were found using cell based assays for testing ligand binding efficiency in T47D and Ba/F3 cell lines. 2 molecules, SMI 1 and SMI 6, were identified as potential inhibitors of the PRLR. Inhibitor cytotoxicity was tested using an LDH assay, utilizing a 468 cancer cell line, to ensure drug viability as a potential treatment. An invasion assay was produced to test cell invasion when exposed to 1nM prolactin in a 468 cell line. Immunoprecipitation and western blotting were used to observe downregulation of the PRLR signaling pathway. Cancer cell proliferation after inhibitor exposure was calculated using flow cytometry.

Results indicated SMI 1 and 6 had minimal toxicity, ensuring both molecules' capabilities to be therapeutic agents. Furthermore, both inhibitors reduced cell invasion, and cell proliferation when exposed to prolactin, compared to exposure to prolactin only. Additionally, the inhibitors also appear to downregulate the STAT‐5 signaling pathway. Thus, both these small molecule antagonists hold great promise in targeting the prolactin receptor as potential cancer therapeutic drugs of the future.

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‐Activity Relationship of Novel Compounds Based on Tamoxifen with Proposed Sigma‐2 Receptor Activity
Authors: Stang Christopher R.T. , Inbody Laura R. , Orahood Cassandra B. , Dick Matt J. , Eichel Amanda M. , Mohamed Ahmed H. , Faler Ashton D. , Zaibek Ann E. , Kinder David H. , Schneider Ryan A.
Published: June 2021

Sigma‐2 receptors are overexpressed in several different types of human cancer cells and are regarded as potential therapeutic targets. Ligands for sigma‐2 receptors have been shown to elicit anticancer properties including growth arrest and apoptosis. While much research has been devoted to designing sigma‐2 receptor ligands for therapeutic benefits, none have stood up to rigorous testing and clinical trials needed for approval in human patients. In an attempt to provide more literature on the potential usefulness of sigma‐2 receptor ligands for anticancer activity, we synthesized a series of analogs based upon the known sigma‐2 receptor ligand tamoxifen that lack estrogen receptor binding properties.

We previously reported on the first molecule in this series, SP‐1, with IC50 activity ranging from 30–60 μM against several dissimilar cell lines. To further elucidate structurally important regions of the molecule and ascertain a structure‐activity relationship (SAR) for anticancer activity, six additional molecules were synthesized and tested. These molecules included modifications to both the terminal phenyl and nitrogen moieties of SP‐1 (N,N‐dimethyl‐2‐(4‐(1‐(p‐tolyl) vinyl)phenoxy)ethan‐1‐amine). The XTT cell viability assay was then used to screen these compounds for anticancer activity in four diverse human cancer cell lines including A‐172 human glioblastoma, A375 human malignant melanoma, HT‐29 human colorectal adenocarcinoma, and MCF7 human breast adenocarcinoma. Interestingly, hydration of the ethylene moiety attached to the carbon linker between the two phenyl rings markedly reduced anticancer activity of SP‐2, SP‐3, SP‐7 and SP‐8. Substantial anticancer activity was not observed at concentrations less than 90 μM regardless of structural modifications made to the periphery of these molecules. However, SP‐5 and SP‐6, which conserved the nonpolar central linker present in SP‐1, demonstrated moderately potent anticancer activity against the four cancer cell lines. SP‐5, which only differs from SP‐1 by the addition of one carbon to the peripheral phenyl ring, was found to have nearly the same potency as SP‐1. However, SP‐6, which includes the addition of a morpholine ring to the periphery of the molecule, demonstrated slightly less potent activity than that of SP‐1 and SP‐5. However IC50 values were still found to range from 45–90 μM in all four cells lines. While the changes made to these analogs failed to improve the efficacy of our parent molecule SP‐1, the information regarding the SAR will help guide the synthesis of further analogs. Additional studies investigating the mechanisms of death and putative targets are ongoing for these lead molecules.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Curcumin Attenuates Expression of Adhesion Molecules by IL‐1 β in HUVECs
Authors: Chang Weng‐Cheng , Yu Ya‐Mei , Cheng An‐Chin
Published: June 2021

Atherosclerosis, a progressive pathological disorder inducing cardiovascular and cerebrovascular diseases, is a leading cause of human morbidity and mortality worldwide. The expression of cell adhesion molecules such as intercellular adhesion molecule (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and adhesion molecule‐1 (E‐selectin) by the endothelium and the attachment of monocytes to endothelium may play a pivotal role in the early atherogenic process. Curcumin is a polyphenol obtained from the root of the turmeric (Curcuma longa), and is the main component of curry powder for giving a specific flavor and yellow color. Previous studies indicated that curcumin exhibits a variety of pharmacological effects including anti‐inflammatory, antitumor, antioxidant, anti‐hypertensive, antiviral, anti‐obesity, anti‐infectious activities and wound‐healing properties. In this study, efforts were made to identify curcumin may aid in the prevention of monocyte‐endothelial cell adhesion induced atherosclerosis. Our results indicated that curcumin could inhibit the expression of adhesion molecules, including ICAM‐1, VCAM‐1, and E‐selectin, and futher decrease the adhesiveness to a human monocytic cell line (U937) in human umbilical vein endothelial cells (HUVECs). These results demonstrated that curcumin has inhibitory effect on proanthersclerotic mechanism in vitro.

Support or Funding Information

This study was supported by NSC 100‐2313‐B‐309‐003 to A‐C Cheng and NSC 95‐2815‐C‐039‐019‐B to Y‐M Yu.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of STAT3 phosphorylation by curcumin partly reduces adhesion molecule expression in vascular endothelial cell
Authors: Ahn Hee Yul , Cho Young Suk , Sankaralingam Sowndramalingam
Published: June 2021

OBJECTIVES

We investigated whether curcumin, an ingredient of turmeric, can suppress the expression of intercellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the underlying mechanisms.

MATERIALS AND METHODOLOGY

The levels of adhesion molecules, activities of nuclear factor kappaB (NF‐kB) and signal transducer and activator of transcription 3 (STAT3) were determined using Western blot analysis. The effect of curcumin on monocyte binding to endothelial cells was determined using THP‐1 monocyte (human acute monocytic leukemia cell line) adhesion assay.

RESULTS

Curcumin prevented the LPS‐mediated increase in ICAM‐1 and VCAM‐1 expression in HUVEC in a dose‐dependent manner. Curcumin did not affect LPS induced degradation of IkappaBα (IkBα). On the other hand, curcumin reduced the LPS‐induced STAT3 phosphorylation. Curcumin significantly reduced LPS‐mediated THP‐1 monocyte adhesion to HUVEC. Furthermore, in C57BL/6 mice, curcumin prevented the LPS induced increase in aortic ICAM‐1 and VCAM‐1 expression.

CONCLUSIONS

Curcumin prevented LPS‐induced ICAM–1 and VCAM–1 expression on vascular endothelial cells associated with inhibition of STAT3 phosphorylation.

Support or Funding Information

This work was supported by the research grants of the Chungbuk National University in 2014.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FAK maintenance of endothelial mechanotransduction controls epigenetic repression of KLF2 and S1PR1 transcription
Authors: Akhter Md Zahid , Yazbeck Pascal , Srivastava Nitya N. , Tauseef Mohammad , Tennes‐Schmidt Tracy , Lenzini Stephen B. , Shin Jae‐Won , Mehta Dolly
Published: June 2021

Sphingosine‐1‐phosphate (S1P) activation of sphingosine‐1‐phosphate receptor 1 (S1PR1) in endothelial cells (ECs) strengthens the endothelial barrier and prevents lung injury. However mechanisms regulating EC‐S1PR1 expression remains unclear. Recently, we showed that endothelial‐specific deletion of focal adhesion kinase (FAK) in mice severely disrupted lung vascular barrier function. Microarray analysis of FAK depleted cells showed a marked reduction in S1PR1 expression. Thus, we tested the hypothesis that impaired S1PR1 synthesis is responsible for defective endothelial barrier repair in EC‐FAK−/− mice. We confirmed that FAK deletion markedly reduced S1PR1 expression both at the mRNA and protein level in lungs and ECs whereas the expression of S1PR2 and S1PR3 and S1P generating enzymes, sphingosine kinases (SPHK1 and SPHK2) was not reduced. Consistently, FAK depleted ECs and mice conditionally lacking FAK in pulmonary ECs failed to anneal adherens junctions and showed impairment of endothelial barrier function under basal condition and even after S1P application. Rescuing S1PR1 expression in FAK null ECs restored barrier function in the pulmonary vessels of EC‐FAK null mice. Next, we screened for potential transcription factors binding to the S1PR1 promoter which included KLF2, Sox2, FoxP1, GATA1, GATA4 and ERG1. Intriguingly, we observed that rescuing KLF2 expression in FAK depleted ECs restored S1PR1 expression and endothelial barrier function to the levels observed in control cells. In other studies, we observed that KLF2 increased S1PR1 promoter activity while the S1PR1 cDNA lacking KLF2 binding sites failed to increase S1PR1 promoter activity, demonstrating that KLF2 can promote endothelial barrier function in the absence of EC‐FAK by transducing S1PR1. Because FAK maintains intracellular tension by balancing the activities of RhoA and Rac1 GTPases and KLF2 mRNA expression is regulated by methylation of CpG islands, we next address the possible role of increased cellular tension and DNA methyltransferases (DNMT) in suppressing KLF2 gene transcription and thereby S1PR1 expression in EC‐FAK null mice. We found that DNMT activity was elevated by 3‐fold in FAK depleted ECs in a RhoA‐dependent manner. Intriguingly, we found that depletion of FAK increased EC‐stiffness, which was associated with hypermethylation of KLF2 by DNMT3a. Interestingly, inhibition of RhoA in FAK depleted cells restored KLF2 and S1PR1 expression. Thus, our data describes the novel role of FAK in suppressing cellular tension driven‐epigenetic modification of KLF2 and S1PR1 and thereby vascular integrity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiac‐specific Overexpression of MiR‐133a Decreases Pyroptosis in Ins2+/− T1DM Mice Heart
Authors: Kambis Tyler N. , Yadav Santosh K. , Mishra Paras K.
Published: June 2021

Increased pyroptosis (a caspase‐1‐dependent cell death) and decreased miR‐133a (the most abundant cardioprotective miRNA in the human heart) are documented in the diabetic mellitus (DM) heart. However, it is unclear whether the reduced miR‐133a in DM hearts induces cardiac pyroptosis. We hypothesized that lack of miR‐133a in the diabetic heart induces pyroptosis, which is suppressed by the overexpression of miR‐133a. To test the hypothesis, we created a novel diabetic mice strain where miR‐133a is overexpressed only in the heart (Akita/miR‐133aTg), by crossbreeding Ins2+/− Akita (spontaneous, genetic T1DM) male with cardiac‐specific miR‐133a transgenic (miR‐133aTg) female mice. We have used sibling Ins2+/+ mice as WT for Ins2+/− Akita mice. In the four groups (WT, Akita, Akita/miR‐133aTg and miR‐133aTg) of fourteen‐week male mice, we measured the levels of pyroptosis markers NLRP3 (involved in the inflammasome complex formation that is required for pyroptosis initiation), caspase‐1 (the key enzyme for pyroptosis), and interleukine‐1β (IL‐1β) (a pro‐inflammatory cytokine that is involved in pyroptosis) in the heart. The densitometric analyses of Western blots revealed that protein levels of pyroptosis markers are upregulated in Akita hearts, whereas the overexpression of miR‐133a blunts this upregulation. The protein expression of different pyroptosis markers in the above‐mentioned four groups of hearts were; NLRP3: WT 0.04 ± 0.0, Akita 0.09 ± 0.1, Akita/miR‐133aTg 0.03 ± 0.0, miR‐133aTg 0.01 ± 0.0; caspase 1: WT 0.7 ± 0.1, Akita 1.0 ± 0.1, Akita/miR‐133aTg 0.2 ± 0.0, miR‐133aTg 0.1 ± 0.0, and IL‐1β: WT 1.9 ± 0.6, Akita 2.1 ± 0.3, Akita/miR‐133aTg 1.1 ± 0.0, miR‐133aTg 0.8 ± 0.1 (the standard deviation values are rounded to one decimal point). In conclusion, this is the first report showing that cardiac‐specific overexpression of miR‐133a mitigates DM‐induced pyroptosis in the heart.

Support or Funding Information

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rbfox1 regulates proteasomal activity and K48‐linked ubiquitination
Authors: Umar Sadiq , vanBerlo Jop
Published: June 2021

Rbfox1 is important to maintain cardiac function through regulation of alternative splicing and expression of calcium handling proteins. The heart recycles proteins and degrades them either via the ubiquitin proteasome system or through autophagy. The role of Rbfox1 in post‐translational modification (PTM) and proteasomal degradation is not known. To assess a potential role for Rbfox1 in regulating PTM and proteasomal degradation, we deleted Rbfox1 in cardiomyocytes, either by Rbfox1 gene‐deletion mice (cRbfox1−/−) or by transfecting cultured neonatal rat cardiomyocytes with Rbfox1siRNA. Cardiomyocyte specific deletion of Rbfox1 was confirmed by Western blotting and immunohistochemistry. cRbfox1−/− mice showed an overall decrease in expression of SUMO‐1 and K63‐ubiquitin conjugated proteins while K48‐Ubiquitin expression was increased. These results were confirmed in cardiomyocytes transfected with Rbfox1 siRNA, suggesting a direct role for Rbfox1 in regulating sumoylation and ubiquitination. Moreover, we measured the 20S proteasome activity and found an increase in proteasomal activity both in cRbfox1−/− mouse as well as in Rbfox1siRNA transfected cardiomyocytes. Since protein abundance is a balance between protein production and degradation, we further investigated protein synthesis. We transfected cardiomyocytes with siRNA against Rbfox1 and treated them with puromycin. Cell lysates were probed with a puromycin antibody to measure overall protein synthesis. Our results showed significant deficiency in total protein synthesis in Rbfox1 siRNA as compared with control. In conclusion, Rbfox1 is important in maintaining protein homeostasis, by controlling their production and degradation. These alterations in critical cellular processes likely predispose the heart to cardiac dysfunction.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional anatomy of the masticatory muscles in a caviomorph rodent, Cavia porcellus
Authors: Druzinsky Robert E. , Ulm Ashley , Vinyard Christopher J. , Iriarte‐Diaz Jose
Published: June 2021

Guinea pigs (Cavia porcellus) and other caviomorph rodents are hystricognaths. In hystricognaths, there is a groove for the pars reflexa of the superficial masseter in the ventral border of the mandible (Druzinsky, 2007). In the guinea pig (C. porcellus) the tendon of the pars reflexa has a dense connective tissue sesamoid that permits the tendon to glide through the groove. These South American hystricognaths are also hystricomorphs. The zygomatico‐mandibularis muscle originates on the snout and converges on a tendon that runs posteriorly through the infraorbital foramen. The tendon runs over the bony root of the zygomatic arch and turns inferiorly, to insert at the anterior end of the masseteric groove. A second sesamoid is found in this tendon, which allows the tendon to glide over the root of the zygomatic arch. Each of these elaborations of the masticatory muscles is a pinnate muscle that curves around a bony structure, acting as a pulley, and permitting the muscle to be longer than similar muscles in other mammals.

To analyze the mechanics of the caviomorph masticatory apparatus we are creating a 3D model of the skull, jaw, and masticatory muscles through contrast enhanced micro‐CT. The head of an adult guinea pig was soaked in a solution of called iodine potassium iodide (I2KI) for several weeks and then scanned at Northeast Ohio College of Medicine using the Scanco vivaCT micro‐CT. The Materialize Mimics® Innovation Suite will be used to segment the bones, teeth, and muscles to capture the detailed morphology. Modeling of length‐tension relationships and mechanical advantage suggests that the pulley mechanisms of the masseter complex permit these muscles to be long, which allows them to have long excursions and produce significant forces at large gapes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enoxaparins Derived from Ovine (Sheep) Tissues are Biosimilar to Branded Enoxaparin of Porcine (Pig) Origin
Authors: Kouta Ahmed , Hoppensteadt Debra , Abro Schuharazad , Clarizio Destini , Jeske Walter , Yao Yiming , Fareed Jawed
Published: June 2021

Background and Purpose

Low molecular weight heparins (LMWH) are widely used for the management of thrombosis. Enoxaparin represents one of the most widely used LMWH, obtained by depolymerizing porcine (pig) mucosal tissues. Recently, heparin from sheep mucosal tissue has also become available which exhibits a comparable anticoagulant profile to porcine heparin. The purpose of this study is to compare enoxaparin from ovine mucosa with branded enoxaparin.

Materials and Methods

Four batches of enoxaparin were commercially obtained (SanofiAventis, Bridgewater, NJ, USA). Four ovine enoxaparins were provided by Ronnsi Pharmaceutical Co., China. These enoxaparins were compared for the anticoagulant (ACT, aPTT, TT), antiprotease (Anti‐Xa, Anti‐IIa), and their potency was cross‐referenced using NIBSC LMWH standard in an amidolytic Anti‐Xa method (Hyphen Biomedical, Paris, France). Molecular profile studies were carried out as specified by U.S. FDA. Protamine titration studies were carried out for both preparations using various tests. The relative interactions of these enoxaparins with HIT antibodies were also investigated using HIT antibodies obtained from patients employing platelet aggregation studies. Thrombin generation inhibition studies were also carried out using a fluorogenic substarte method (Technoclone, Austria)

Results

The molecular profile of ovine and porcine enoxaparins were comparable and ranged from 4.2–4.5 kDA (p<0.05). The anticoagulant activities in all the assays were also comparable for the ovine and porcine enoxaparins. In the anti‐Xa assays, the NIBSC cross‐referenced potency of several batches of porcine enoxaparin ranged from 98–110 U/mg (mean=102+/−4) whereas the ovine enoxaparin exhibited 102–112U/mg (mean=106+/−6) potency (p<0.05). In the anti‐II assays the potency of the ovine enoxaparins ranged from 25–30 u/ml (mean 28 ± 3) and porcine enoxaparin were 28–35 (mean 31 ± 3). In the protamine titration curves, both enoxaparin preparations provided superimposable results. Similarly, in the HIT antibody assays, the behavior of ovine and porcine enoxaparin was identical.

Conclusions

These studies demonstrated that ovine mucosal tissue‐derived enoxaparin exhibits similar molecular and anticoagulant profiles. In the NIBSC cross‐referenced potency assay, the potency of these preparations were similar. Thus, ovine enoxaparins exhibit comparable profiles when compared to their porcine counterparts and are expected to provide similar results in various clinical indications.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alkaloid Fraction of Alstonia Boonei Stem Bark Demonstrate Antiobesity Potential on High Fat Diet Induced Obesity in Male Sprague‐Dawley Rats
Authors: Anyanwu Gabriel Oluwabunmi , Onyeneke Chukwu Eusebius , Iqbal Jamshed , Qamar Misbah ud Din , Khan Shafi Ullah , Rehman Sabi ur , Rauf Khalid , Rehman Nisar ur
Published: June 2021

The aim of this study was to investigate the anti‐obesity potential of alkaloid fraction of Alstonia boonei stem bark on high fat diet (HFD) induced obesity in male Sprague‐dawley rats.

Preparation of neutral alkaloid fraction (NAF) was done through solvent partitioning of the methanol extract of Alstonia boonei stem bark, and NAF was subjected to LCMS‐MS analysis. To induce obesity, rats were fed with HFD (60% fat content) for 12 weeks preceding oral treatment with 125 and 250 mg/kg body weight of NAF for 4 weeks. Experimental procedures involving rats followed the European Community guidelines (EEC Directive of 1986; 86/609/EEC) for animal use.

The major compounds found in NAF includes: Ajmalicine, 16‐Methoxy‐2,3‐dihydro‐3‐hydroxytabersonine, Vincamine, Aspidofractine, 3‐Hydroxyquinidine, 16‐Methoxytabersonine, Mitraphylline, 18‐Hydroxyyohimbine and Catharanthine. NAF significantly (p < 0.05) decreased body weight, white adipose tissue, triglycerides, total cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, leptin, insulin and dopamine levels, but increased significantly (p < 0.05) the high density lipoprotein cholesterol, cortisol, serotonin and noradrenaline levels in the treated groups compared to HFD control. However, there were no significant (p > 0.05) changes in food intake, blood glucose, thyroxine, triiodothyronine and thyroid stimulating hormone levels in animals given NAF when compared to HFD control. The photomicrograph of liver of the rats treated with NAF showed slight fat changes unlike that of the HFD obese controls which showed presence of numerous prominent fat deposits on the livers.

The findings in this study suggests that the alkaloid fraction of A. boonei possess antiobesity properties as indicated in improved lipid profile, leptin sensitivity, and decreased body weight of animals.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Methanol Extracts from Roots, Leaves, and Fruits of the Lebanese Strawberry Tree (Arbutus andrachne) on Cardiac Function Together with their Antioxidant Activity
Authors: Abidi Emna , Habib Jean , Chahine Nathalie , Mahjoub Touhami , Elkak Assem
Published: June 2021

CONTEXT

Several plant‐derived natural products have been used in clinical phase for applications in neurological, cardiovascular, and inflammatory diseases. Arbutus andrachne L. (Ericacea) is an evergreen shrub native to the Mediterranean region. Traditionally, the fruits and leaves of Arbutus tree are well known and used as antiseptics, diuretics, blood tonic, and laxatives.

OBJECTIVE

Data regarding the biological effects of compounds derived from the Lebanese Arbutus andrachne are not available. In the present work, we studied the antioxidant activity of methanol extracts of leaves, fruits, and roots of the plant against electrolysis; together with their effects on the cardiodynamics of isolated perfused rabbit hearts.

MATERIALS AND METHODS

In vitro electrolysis of the different root, leaves, and fruits methanol extracts was evaluated by the amount of free radicals that has been reduced by increasing the concentration of root extracts ranging from 0.5 to 2 mg after 1, 2, 3, and 4 min. Left ventricular pressure (LVP), heart rate (HR), and coronary flow (CR) were investigated in isolated rabbit heart after administration of 0.5, 1, 2, and 2 mg of each methanol extracts plotted against time (0, 0.5, 1.5, 5, and 10 min), according to the Langendorff method. Lipid peroxidation study was performed by the colorimetric method on myocard tissue after incubation with 500 μl of the different methanol extracts. The amount of MDA was determined at 500 nm absorbance after 5 min incubation.

RESULTS

Among the different methanol extracts, the roots showed the highest in vitro antioxidant activity, particularly observed at concentration of 2 mg which completely inhibits free radical generation after 4 min. LVP decreases by 32% at the dose of 2 mg of root extracts after 5 min. No significant effect was observed by the three tested extracts on the heart rate. The three methanol extracts did not show any significant effect on the coronary flow. Moreover, the roots show an increase in the coronary flow at a concentration of 1 and 2 mg/ml during 1 min. Electrolysis on heart tissue treated with the roots extracts shows a decrease in the MDA level from 70.51 ± 6.71 to 48.58 ± 4.15 nmole/g of tissue.

DISCUSSION AND CONCLUSION

Methanol extracts of the roots possess antihypertensive effect that may result from its ability to decrease the LVP together with its protective role by inhibiting free radical generation and significantly decreasing the MDA level of heart tissue.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Applied Predictive Modeling of Coronary Microvascular Disease using Coronary Doppler and Cardiac Echocardiography
Authors: Patel Kishan U. , Sunyecz Ian L. , McCallinhart Patricia E. , Bartlett Christopher W. , Trask Aaron J.
Published: June 2021

Introduction

Type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) show early inward hypertrophic remodeling and reduced wall stiffness, which are associated with decreased blood flow (CBF), coronary flow reserve, and disrupted flow patterns. These data may suggest that coronary microvascular disease (CMD) underlies the early pathophysiology of T2DM. Furthermore, distinct correlations have been identified between the Doppler coronary flow pattern in both normal and T2DM mice, potentially allowing for a predictive relationship between coronary flow patterns and CMD. Currently, CMD is extremely difficult to diagnose due to lack of non‐invasive methods. The goal of this study was to develop an applied predictive model for CMD non‐invasively in early T2DM using CBF combined with functional and structural measurements of the heart.

Methods

CBF of the left main coronary artery, aortic flow, E & A wave, left ventricular structural dimensions, and aortic diameter were recorded using high frequency, high resolution non‐invasive Doppler echocardiography (Vevo2100, Visual Sonics, Toronto, Canada) in 37 normal heterozygous Db/db mice and 36 T2DM homozygous db/db mice. Coronary flow patterns were analyzed using a Matlab program developed in our laboratory to identify 13 distinct parameters. All other flow patterns and structural and functional features were manually measured in the Vevo2100 software resulting in 13 additional parameters. All the parameters were feature engineered, and a factor analysis model was created and tested using various machine learning algorithms.

Results

Physiological data were subjected to a variety of machine learning models, and the “glmnet algorithm” in R software (generalized linear regression via penalized maximum likelihood) best predicted CMD with a cross validation accuracy of 80.19% and test dataset accuracy to date of 84.84%.

Conclusion

An applied predictive model for CMD in T2DM was developed using 73 mice, which shows a promising ability to accurately predict CMD in T2DM. Further studies will be undertaken to improve the predictive accuracy of the model to be suitable for clinical diagnostics and/or decision support.

Support or Funding Information

Supported by the National Institutes of Health (R00 HL‐116769 to AJT) and Nationwide Children's Hospital (to AJT and CWB).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blood Pressure and Vascular Function in Hypertensive Individuals: Partitioning cause and effect
Authors: La Salle D. Taylor , Broxterman Ryan M. , Ratchford Stephen M. , Richardson Russell S. , Trinity Joel D.
Published: June 2021

Introduction

Vascular dysfunction is a hallmark of hypertension and contributes to an increased risk of cardiovascular disease. Pharmacological interventions to treat hypertension generally result in improved blood pressure status and concomitant improvements in vascular function. However, recent findings have challenged this notion as vascular dysfunction appears to persist despite blood pressure control with antihypertensive medications. Importantly, medications differ in their vasoactive properties and may have direct effects on the vasculature independent of their impact on blood pressure. Therefore, it is difficult to discern if the potential improvements in vascular function are associated with lower blood pressure or a direct effect on vascular function. Utilizing the dietary approach of salt restriction this study examined the impact of reducing blood pressure on vascular function and peripheral hemodynamics in individuals with essential hypertension.

Methods

Hypertensive adults (male n=14, female n=8, 50±10 yr) completed two dietary phases: liberal sodium (LS) (200 mmol/d) followed by restricted sodium (RS) (10 mmol/day) diet for five days, respectively. Antihypertensive medications were withdrawn 2 weeks before the first intervention. Following each dietary phase, measurements of systolic (SBP), diastolic (DBP), and mean arterial blood pressure (MAP), arterial diameter and blood velocity (Doppler ultrasound) were assessed at rest, during brachial artery (BA) flow mediated dilation (FMD), and during progressive handgrip (HG) exercise performed at 15, 30 and 45% of maximal voluntary contraction (MVC).

Results

RS significantly lowered SBP (LS: 140±3, RS: 123±3 mmHg), DBP (LS: 85±2, RS: 79±2 mmHg) and MAP (LS: 103±2, RS: 94±2 mmHg) (all p<0.05). Despite improvements in blood pressure, conduit artery function, as measured by FMD, was similar between conditions (LS: 4.6±0.4%; RS: 5.1±0.5%). Similarly, the hyperemic response during FMD, an assessment of microvascular function, was not different between conditions (LS: 548±43; RS: 615±44 ml). During HG exercise, MAP was lower at all exercise intensities during RS compared to LS (15% MVC; LS: 109±2 vs. RS: 102±2 mmHg; 30% MVC; LS: 113±2 vs. RS: 105±2 mmHg; 45% MVC; LS: 122±3 vs. RS: 112±3 mmHg) while vascular conductance and BA vasodilation were not different between LS and RS (p>0.05).

Conclusion

Despite improvements in blood pressure at rest and during exercise, both conduit artery and microvascular function measured during FMD and HG exercise‐induced vasodilation were not improved. These findings support recent evidence that vascular dysfunction persists in individuals with hypertension independent of blood pressure status.

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