bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐Palmitoylethanolamide‐oxazoline Treatments Protects Against Neuroinflammatory Injury Induced by Transient Middle Cerebral Artery Occlusion in Diabetic Rats
Authors: Di Paola Rosanna , Fusco Roberta , Cordaro Marika , D'Amico Ramona , Gugliandolo Enrico , Siracusa Rosalba , Peritore Alessio , Crupi Rosalia , Impellizzeri Daniela , Cuzzocrea Salvatore
Published: June 2021

Diabetes causes various macrovascular and microvascular alterations often culminating in major clinical complications, first of all is stroke, that, until today, lacks effective therapeutic intervention. N‐Palmitoylethanolamine‐oxazoline (PEA‐OXA) possesses anti‐inflammatory and potent neuroprotective effects. Although, recent studies have explained the neuroprotective properties of PEA‐OXA, nothing is known about its effects in treating cerebral ischemia. The aim of this study is to explore the mechanism and the effect of PEA‐OXA against cerebral ischemic/reperfusion injury using the transient middle cerebral artery occlusion (MCAo) model in diabetic rats. Neurological severity score and infarct volumes were carried out to assess the neuroprotective effects of PEA‐OXA. Moreover we observed PEA‐OXA‐mediated improvements in tissues histology shown by reduction of lesion size and improvement in apoptosis level (assessed by Caspases, Bax and Bcl‐2 modulation and TUNEL assay) further support the efficacy of PEA‐OXA therapy. We also found that PEA‐OXA treatment was able to reduce mast cell degranulation and to block infiltration of astrocytes and microglia and reduced MCAo‐induced expression of cytokines, nitrotyrosine, MDA, VEGF and TGF‐b, as well as was competent to re‐establish growth factors such as BDNF, GDNF and NGF. PEA‐OXA also inhibited the MCAo‐mediated increased expression of NAAA and NF‐κB and degradation of IκB‐α. PEA‐OXA treated injured animals also improved neurobehavioral functions as evaluated by water maze test. Based on these findings, we propose that PEA‐OXA would be useful in decreasing the risk of impairment or improving function in ischemia‐reperfusion brain injury‐related disorders.

Support or Funding Information

no funding

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium‐activated K+ channel KCa3.1 Plays a Pro‐inflammatory Role in α‐Synuclein Models of Parkinson's Disease
Authors: Malovic Emir , Sarkar Souvarish , Nguyen Hai , Singh Vikrant , Jin Huajun , Anantharam Vellareddy , Kanthasamy Arthi , Wulff Heike , Kanthasamy Anumantha G.
Published: June 2021

Chronic neuroinflammation characterized by the sustained activation of microglia and subsequent release of pro‐inflammatory mediators is concomitant with the progressive neurodegeneration observed in Parkinson's disease (PD). Microglial potassium channels have a well‐developed pharmacology and constitute attractive targets for reducing neuroinflammation. We previously implicated the voltage‐gated channel Kv1.3 in playing a proinflammatory role in cell culture and animal models of PD. Another K+ channel, the calcium activated channel KCa3.1 is also expressed in a variety of immune cells, including B cells, T cells, and microglia. However, the role of KCa3.1 in PD‐related neuroinflammatory processes has not been investigated. In this study, we examined whether KCa3.1 plays a role in regulating neuroinflammation in PD. Exposure of primary mouse microglia to aggregated α‐synuclein (α‐Synagg) led to the upregulation of KCa3.1 mRNA, as determined by qRT‐PCR. Patch‐clamp recordings revealed that KCa3.1 activity is also upregulated following α‐Synagg stimulation. We further demonstrated that α‐Synagg increases the production of pro‐inflammatory cytokines, including IL‐1β, IL‐6, IL‐12, and TNFα, while these cytokines were significantly attenuated with the KCa3.1 inhibitor, TRAM‐34 (1‐[(2‐chlorophenyl)diphenylmethyl]‐1H‐pyrazole). Using proteomics analysis, we identified potential targets that may be involved in KCa3.1 signaling in α‐Synagg‐treated samples. In animal studies, C57BL/6J mice received a unilateral stereotaxic injection of mouse α‐synuclein preformed fibrils (α‐Synpff) in the striatum. One subset of these animals received TRAM‐34 (10 mg/kg, i.p.) daily starting at 4 months and animals were sacrificed one month later. Behavioral analysis revealed α‐Synpff‐induced motor deficits at 5 months post‐injection, whereas TRAM‐34 attenuated α‐Synpff‐induced motor deficits. TRAM‐34 was also able to significantly block α‐Synpff‐induced decreases in striatal dopamine and DOPAC levels. This was further corroborated by TRAM‐34's protection against the α‐Synpff‐induced loss of tyrosine hydroxylase in both substantia nigral and striatal tissues by immunohistochemistry and immunoblotting, respectively. Collectively, our data highlight the pivotal role of KCa3.1 in PD‐associated neuroinflammatory processes and has potential therapeutic implications.

Support or Funding Information

(NIH grants NS100090, NS088206 and ES026892)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of matirx‐metalloproteinases mediates carnosine‐neuroprotection during ischemia
Authors: Kim Eun‐Hye , Shin Donggeun , Shin Young‐Jun , Kim Donghyun , Oh Eujin , Majid Arshad , Bae Ok‐Nam
Published: June 2021

Despite the increasing global medical burden of ischemic stroke, there is no existing therapeutic option against ischemic stroke. It is urgent to find a new therapeutic candidates or therapeutic targets for ischemic stroke. Matrix‐metalloproteinases (MMPs) contribute to deterioration of neuronal damage by breakdown of blood‐brain barrier and edema formation during ischemic stroke, and inhibition of MMP activity is known to reduce ischemic brain injury. Our group recently showed that the endogenous dipeptide, carnosine, has a high protective activity against ischemic brain damage, suggesting that carnosine may be a new candidate for stroke treatment. In this study, we investigated whether carnosine regulates the activity of matrix‐metalloproteinases (MMPs) and brain edema by in vivo rat stroke models and in vitro enzyme activity assay. Carnosine therapy has significantly reduced neuronal infarct as well as edema in rats with ischemic stroke initiated by permanent occlusion of middle cerebra artery. Inhibitory effect of MMP activation by carnosine were observed in gelatin zymography and western blot of brain hemispheres. In the isolated enzyme assay, carnosine significantly inhibited the activity of MMP‐2 and MMP‐9 mediated by chelation of zinc, which is essential for the MMP activity. Taken together, we have found that carnosine can inhibit MMP activity, which reduces edema formation and brain damage by reducing the level of co‐factor, zinc needed. We believe that our research provides new insights into the carnosine neuroprotection mechanism of ischemic brain injury.

Support or Funding Information

Funding Source: This work was supported by grants from the National Research Foundation (NRF‐2017R1C1B3002626) of Korea.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Guanabenz Modulates Microglia and Macrophages in EAE
Authors: Thompson Kaitlyn Koenig , Tsirka Stella E.
Published: June 2021

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by peripheral immune cell infiltration, demyelination, and axonal damage. Microglial activation has also long been recognized as an early event in MS pathogenesis and is generally considered harmful as it can contribute to pro‐inflammatory responses and neuronal damage. However, microglia are also capable of releasing factors that can suppress the immune response and promote remyelination and repair. Therefore, a valid therapeutic strategy is to prevent pro‐inflammatory microglial activation and/or shift their phenotype towards neuroprotective and anti‐inflammatory. We have previously characterized the immunomodulatory tetrapeptide, tuftsin, that attenuates the experimental autoimmune encephalomyelitis (EAE) model of MS by inducing an anti‐inflammatory shift in microglia and infiltrating monocyte‐derived macrophages. As tuftsin is not FDA‐approved, we sought to investigate the repurposing of an already approved drug that may act similarly. We identified guanabenz, an FDA‐approved antihypertensive shown to possess anti‐inflammatory properties, using a virtual screen against tuftsin's receptor, neuropilin‐1 (NRP1). Although guanabenz does not bind NRP1, it does provide beneficial anti‐inflammatory effects in EAE. Using two EAE models that represent chronic and relapsing‐remitting (RR) MS disease courses we observe that guanabenz has beneficial, microglia/macrophage‐targeted immunomodulatory properties. In the chronic EAE model guanabenz improves disease scores, reduces demyelination and the amount of harmful, pro‐inflammatory microglia when administered therapeutically. In the RR‐EAE model guanabenz attenuates relapses, reduces demyelination and pro‐inflammatory microglia numbers when administered immediately after the initial symptom peak. Expression of pro‐inflammatory genes, such as TNF‐a, is reduced in stimulated primary macrophages and microglia. Overall, we report that guanabenz is capable of dampening the pro‐inflammatory responses of microglia and macrophages and improving pathological hallmarks and motor deficits in EAE. Further, combination of guanabenz with clemastine, an FDA‐approved antihistamine now in clinical trials for inducing remyelination in MS, offers additional benefits.

Support or Funding Information

This work was supported by NMSS CA1044A1 and PP1815 (SET) and PhRMA predoctoral fellowship (KKT).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The anti‐glioblastoma drug ONC201 and related analogs act as non‐competitive antagonists at the D2 dopamine receptor through unique binding site interactions
Authors: Cuoco Caroline Ann , Free R. Benjamin , Willette Blair , Lane J. Robert , Allen Joshua E. , Sibley David R.
Published: June 2021

ONC201 is a first‐in‐class imipridone molecule that is in clinical trials for the treatment of glioblastoma. It was initially identified as a p53‐independent inducer of the immune cytokine TRAIL leading to tumor cell death. Notably, overexpression of the D2 dopamine receptor (D2R) occurs in a variety of cancers, including glioblastoma, and its antagonism imparts pro‐apoptotic and anti‐proliferative effects in tumor cells. Characterization of ONC201 and several novel analogs (ONC206 and ONC213) using functional assays (β‐arrestin recruitment and cAMP accumulation) and radioligand binding analyses revealed that all ONC compounds were full antagonists of the D2R with low micromolar affinities, similar to their potencies for anti‐proliferative effects. To investigate the mechanism of D2R antagonism by the ONC compounds, Schild‐like curve‐shift experiments were performed using the functional assays where dopamine dose‐response curves were constructed in the presence of increasing concentrations of ONC compound. The addition of ONC compounds caused both a rightward shift in the dopamine concentration‐response curve and a depression of the maximum response. Such results are consistent with a mixed/non‐competitive mechanism of antagonism. These curve shift data were fit to the operational model of allostery, which revealed that the predominant modulatory effect of the ONC compounds is on dopamine efficacy with a smaller effect on dopamine potency. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis where amino acids are systematically mutated to alanine coupled with a D2R‐mediated calcium efflux assay. In this fashion, eight residues, located in two divergent pockets were identified as being important for ONC201's functional antagonism of the D2R and postulated to represent potential ligand‐receptor interaction sites. D2Rs harboring these mutations were expressed in HEK293 cells and assayed for both binding affinity and functional efficacy of the ONC compounds. The ability of ONC201 and analogs to block dopamine‐mediated G protein responses (cAMP and Go‐activation) and to bind to the receptor were hindered by the introduction of each mutant supporting the importance of these interaction sites. Of particular note is the loss of ONC binding affinity and functional efficacy with the distant I397A and V91A mutations possibly suggesting allosteric coupling between these sites. Elucidation of the pharmacological profile and unique interaction sites of ONC201 and analogs may lead to a better understanding of its mechanism for anticancer targeting.

Support or Funding Information

NINDS Intramural Research Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NLRP3 Inflammasomes Contribute to the Impaired Bladder Contraction in Male Diabetic Mice
Authors: Priviero Fernanda , Onuoha Nina , Calmasini Fabiano Beraldi , Ogbi Safia , Webb R. Clinton
Published: June 2021

Background

NLRP3 inflammasomes activation has been demonstrated in both type 1 and type 2 diabetes mellitus and it has been correlated with dysfunctional organs. Although it has been shown that activation of NLRP3 inflammasomes contributes to bladder dysfunction in models of cystitis and bladder outlet obstruction, it is still unknown whether inflammasomes contribute to the development of diabetic bladder dysfunction.

Aim

We aimed to evaluate the contribution of NLRP3 inflammasomes in the late phase of diabetic bladder dysfunction which is known by hypo responsiveness of the detrusor smooth muscle.

Methods

Strips of bladder were excised from male and female wild type (WT) and dbdb−/− mice at 14 – 16 week old and were mounted in a muscle strip myograph system. Contraction of the bladder was evaluated by a single concentration of KCl (120 mM), concentration‐response curves to carbachol (1 nM – 30 μM) and frequency‐response curves to electrical field stimulation (EFS: 1–16 Hz). Carbachol and EFS were repeated in the presence of the NLRP3 inhibitor MCC950 (1 μM).

Results

Male bladder strips presented higher magnitude of contraction than female bladder strips for all contractile agents tested. KCl‐induced contraction was similar in both strains (Female WT: 42 ± 7 mN; dbdb−/−: 40 ± 4 mN; Male WT: 54 ± 6.5 mN; dbdb−/−: 51 ± 17 mN). In males, the potency of carbachol was lower in the bladder of dbdb−/− mice (pEC50: 5.53 ± 0.09) compared to WT (pEC50: 6.11 ± 0.07) and it was not restored in the presence of MCC950 (pEC50: 5.66 ± 0.08). Similarly, the contraction induced by EFS was significantly reduced for all frequencies in the bladder of male dbdb−/− mice (at 16 Hz: 51 ± 10 mN) compared to WT (at 16 Hz: 77 ± 3 mN). However, it was restored in the presence of MCC950 (at 16 Hz: 71 ± 11 mN). In female mice, there was no difference in the contraction of the bladder of dbdb−/− mice when compared to WT and addition of MCC950 did not altered the responses neither in WT nor in dbdb−/−.

Conclusion

In conclusion, our data suggest that NLRP3 inflammasomes contribute to the development of hypoactive detrusor response in the late phase of diabetic bladder dysfunction in males.

Support or Funding Information

National Institute of Health (NIH; HL‐134604) São Paulo Research Foundation (FAPESP; 2016/20592‐8)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of chronic arsenic oral exposure on hepatic and intestinal CYP expression
Authors: Fan Xiaoyu , Liu Pengfei , Han Weiguo , Wu Xiangmeng , Ding Liang , Zhang Donna , Ding Xinxin , Zhang Qing‐Yu
Published: June 2021

Environmental exposure to arsenic is associated with increased incidences of multiple adverse health consequences in humans, posing a significant health risk to people living near contaminated sites. For example, populations residing near mining sites may be exposed to mine tailings through the inhalation of dust particles or ingestion of contaminated water or food, as the arsenic contaminants may become bio‐accessible. Previous studies in rodents reported the ability of short‐term exposures to arsenic at high doses to increase or decrease the expression of several xenobiotic metabolizing CYPs in liver, lung, heart, or kidney, raising the possibility that exposed human individuals may have altered ability to metabolize drugs and other xenobiotic compounds. The aim of this study is to examine effects of chronic arsenic oral exposure at a relatively low level on expression of various CYPs in the liver. In the first experiment, mice were fed control or sodium arsenite‐containing drinking water (25 ppm) for 20 weeks. Hepatic gene expression was analyzed using RNA‐seq, which revealed significant increases in transcript levels for several CYPs, including CYP2A5 (by 10‐fold), an enzyme known for its activities toward nicotine, various environmental toxicants, as well as endogenous compounds. In a follow‐up study, hepatic CYP2A5 transcript level, determined by real‐time PCR, was also found to be increased (by 2.5‐fold) after 4 weeks of exposure to arsenic. Induction of CYP2A protein was confirmed by immunoblot analysis of both sets of samples. Like CYP2A5, hepatic expression of CYP3A11, a major drug‐metabolizing CYP enzyme, was also induced by arsenic exposure for 4 or 20 weeks. CYP expression in the intestine was also examined in the 4‐week exposed groups. CYP3A11, but not CYP2A5, is expressed in the intestine. Induction of CYP3A11 expression was observed in the proximal but not distal intestine, and it was less than 2‐fold. Taken together, these data reveal significant induction of hepatic CYP2A5 and CYP3A11 expression by chronic exposure to a low dose of arsenic in drinking water. The impact of this model of arsenic exposure on hepatic and intestinal xenobiotic metabolism is currently under study.

Support or Funding Information

NIH grants ES020867 and GM082978

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Dual Farnesoid X Receptor Agonist /Soluble Epoxide Hydrolase Inhibitor Prevents Non‐Alcoholic Steatohepatitis in Mice
Authors: Khan Abdul Hye , Stavniichuk Anna , Schmidt Jurema , Merk Daniel , Imig John D
Published: June 2021

Non‐alcoholic steatohepatitis (NASH) is becoming the most prevalent metabolic liver disorder and is a serious global health burden. Farnesoid X receptor (FXR) agonists are in late stage clinical development to counter the disease and promise therapeutic efficacy. However, the multi‐factorial nature of NASH pathogenesis and progression indicate the need for a therapeutic approach that will target multiple pathophysiological aspects of NASH. We have developed a dual FXR agonist/soluble epoxide hydrolase (sEH) inhibitor (FXRA/sEHI) and investigated its ability to prevent NASH in toxin‐ and diet‐induced mouse NASH models. Toxin‐induced NASH was achieved in male C57BL/6 mice by injecting 200 μg streptozotocin (STZ) solution two days after birth and feeding a high‐fat diet after 4 weeks of age. Diet‐induced NASH was carried out in adult (8–12 weeks old) male C57BL/6J mice fed a methionine‐choline deficient diet (MCD; D518810) and was compared to mice fed a composition‐matched methionine‐choline replete control diet (D518754) for 4 weeks. In toxin‐induced NASH model, mice were orally treated with vehicle or FXRA/sEHI (10 mg/kg, thrice daily) or an FXR agonist obeticholic acid (OCA, 10 mg/kg, once daily) for 4 weeks. The mice from diet‐induced NASH model treated with FXRA/sEHI (10 mg/kg/d) or vehicle given in drinking water for 4 weeks. At the end of the 4‐week treatment period, blood was collected for biochemical analysis, liver tissue was collected for mRNA expression and histopathological analysis. Mice of the toxin‐induced NASH model had higher liver enzyme, hyperlipidemia, liver fibrosis, and steatosis. FXRA/sEHI treatment reduced these parameters (by 20–50%, P<0.05), while OCA displayed weak or no beneficial effect on NASH (P>0.05). In diet‐induced NASH model, the mice exhibited 2‐fold higher hepatic fibrosis, 2–6‐fold higher hepatic mRNA expressions of inflammatory and fibrotic markers, and marked hepatic steatosis (P<0.05). The dual modulator FXRA/sEHI robustly prevented hepatic steatosis, fibrosis, and inflammation (by 50–80%, P<0.05) compared to vehicle. Our results demonstrate strong potential for dual FXRA/sEHI modulators as a novel therapeutic strategy in NASH and advocate multi‐targeted approaches in NASH treatment.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact on acetaminophen‐induced hepatotoxicity by long non‐coding RNAs HNF1α‐AS1 and HNF4α‐AS1 in HepaRG cells
Authors: Chen Liming , Manautou Jose E. , Zhong Xiao‐bo
Published: June 2021

Acetaminophen (APAP) is a commonly used over‐the‐counter drug for its analgesic and antipyretic effects. However, APAP overdose leads to severe hepatotoxicity due to the accumulation of N‐acetyl‐p‐benzoquinone imine (NAPQI), the toxic metabolite of APAP generated by cytochrome P450s (P450s). Long non‐coding RNAs HNF1α‐antisense 1 (HNF1α‐AS1) and HNF4α‐antisense 1 (HNF4α‐AS1) are regulatory RNAs, which are recently identified to be involved in the regulation of hepatic P450 expression. This study aims to determine whether HNF1α‐AS1 and HNF4α‐AS1 have impacts on APAP‐induced hepatotoxicity using the most useful hepatic cell line HepaRG as an in vitro model. Small hairpin RNAs (shRNAs) were used to knock down HNF1α‐AS1 and HNF4α‐AS1 in HepaRG cells. Knocking down of these lncRNAs altered APAP‐induced hepatotoxicity indicated by MTT and LDH cytotoxicity assays. Specifically, HNF1α‐AS1 knockdown decreased APAP toxicity at 10 mM with increased cell viability and decreased LDH release, whereas HNF4α‐AS1 knockdown exacerbated APAP toxicity at 30 mM. Alterations of gene expression by knocking down HNF1α‐AS1 and HNF4α‐AS1 were examined in several APAP‐induced hepatotoxicity pathways, including P450‐mediated APAP biotransformation by CYP1A2, 2E1, and 3A4, APAP detoxification with glucuronidation by UDP‐glucuronosyltransferases, UGT1A1 and 1A9, and sulfation by sulfotransferases SULT1A1, and NAPQI detoxification through glutathione conjugation by glutathione‐S‐transferases, GSTP1 and GSTT1. HNF1α‐AS1 knockdown significantly decreased mRNA levels of CYP1A2, 2E1, and 3A4, while HNF4α‐AS1 knockdown significantly induced mRNA levels of CYP2E1 and 3A4 in HepaRG cells. HNF1α‐AS1 knockdown decreased mRNA expression of SULT1A1 but not UGT1A1 or 1A9, while HNF4α‐AS1 knockdown showed no significant effect on the selected phase II enzymes. Significantly decreased mRNA expression of GSTP1 was observed after either HNF1α‐AS1 or HNF4α‐AS1 knockdown. However, considering the low intrinsic expression of GSTP1 in HepaRG cells, how this decreased expression of GSTP1 affects APAP toxicity is inconclusive. GSTT1 expression showed no change in all groups. Altogether, our study suggests that HNF1α‐AS1 and HNF4α‐AS1 affect APAP‐induced hepatotoxicity mainly through alterations of P450‐mediated APAP biotransformation process in HepaRG cells, indicating an important role of lncRNAs in drug‐induced liver toxicity.

Support or Funding Information

This work was supported by grant NIH/NIGMS R01GM118367.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and potential involvement of PMAT in mIBG transport in neuroblastoma
Authors: Zhang Yuchen , Quiñones Antonio López , Stevens Jeffrey , Park Julie , Wang Joanne
Published: June 2021

Neuroblastoma (NB) is the most common extracranial solid tumor in pediatric cancer. Despite recent improvements, the long‐term survival rate for patients with high‐risk NB is still less than 50%. Radiolabeled meta‐iodobenzylguanidine (mIBG), 123I‐mIBG and 131I‐mIBG, are used clinically for both diagnostic and therapeutic purposes, and several clinical trials are ongoing to investigate the potential of 131I‐mIBG as a frontline therapy for high‐risk NB. Previous studies established that mIBG enters NB cells through the norepinephrine transporter (NET). However, not all NET‐expressing NBs respond to 131I‐mIBG treatment and other mechanisms may be involved in the uptake and retention of mIBG in NB. Here, we investigated the expression and potential influence of additional mIBG transporters in NB. By searching through an NCI database with 156 NB patients, mRNA Seq results showed a high and variable mRNA expression of the plasma membrane monoamine transporter (PMAT) in NB tumor samples. Interestingly, Kaplan–Meier estimation suggested an association of low PMAT expression with poor survival rates in these patients. Using qPCR analysis, we further demonstrated a high expression of PMAT in 32 local NB samples as well as two human NB cell lines SH‐SY5Y and SK‐N‐BE(2). Immunofluorescence imaging of PMAT in NB tumor specimens and cultured cells suggested that unlike its normal expression on plasma membrane, PMAT protein in NB cells is predominantly expressed in intracellular organelles but also with a low expression at the plasma membrane. In HEK293 cells stably expressing human PMAT, mIBG is avidly transported by PMAT with a Km value of 42.0 ± 10.2 μM. In SH‐SY5Y and SK‐N‐BE(2) cells, uptake of mIBG was performed with or without sodium in uptake buffer to distinguish the NET‐mediated uptake. Preliminary data showed that sodium depletion only inhibited 50–60% of mIBG uptake whereas addition of a PMAT inhibitor to sodium‐free buffer completely inhibited mIBG accumulation in both cell lines. In summary, our current study suggests that PMAT is highly expressed in NB tumors and NB‐derived cells lines. PMAT transports mIBG and may be involved in the uptake and retention of mIBG in NB cells, and hence may represent a novel determinant of mIBG tumor targeting and clinical response.

Support or Funding Information

This work is supported by NIH Grants GM066233 and GM066233‐14S1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Model for Studying Nucleoside Transporters, a Key Component of the Blood‐Testis Barrier
Authors: Miller Siennah R , Morales Mark N , Frost Kayla L , Cherrington Nathan J , Wright Stephen H
Published: June 2021

Sertoli cells in the testis represent the principle element of the Blood‐Testis Barrier (BTB). Equilibrative nucleoside transporters (ENTs) are responsible for the transport of nucleosides across rodent BTB and immunohistochemistry suggests that hENT1 is located on the basal membrane and hENT2 is located on the apicolateral membrane of Sertoli cells (Klein et al., 2013). Therefore, the ENTs are of particular interest in studying the disposition of nucleoside reverse transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in chemical structure to nucleosides. This study characterized the transport of a specific nucleoside, uridine, to determine the relative roles of ENT1 and ENT2 in nucleoside transport in HeLa S3 cells, a potential model system for the study of ENT selectivity, using the ENT specific inhibitor S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR). Uptake of 20 nM [3H]uridine was linear for 7 minutes and 100 nM NBMPR blocked 77% of this uptake, with the remaining uptake blocked by 100 uM NBMPR (to the same level produced by 5 mM unlabeled uridine). ENT1 and ENT2 are known to have different sensitivities for NBMPR and, consistent with this difference, inhibition of [3H]uridine uptake produced by increasing concentration of NBMPR proved to be biphasic, with IC50s of 11.1 nM (ENT1) and 7.2 uM (ENT2). Consequently, uridine uptake measured in the presence of 100 nM NBMPR was taken to represent ENT2‐mediated transport; subtracting that from total uptake represented ENT1‐mediated transport. The kinetics of ENT1‐ and ENT2‐mediated [3H]uridine uptake into HeLa S3 cells showed no difference in Jmax (16.1 and 12.3 fmol cm−2 min−1) and a three‐fold difference in Km (13.3 and 35.2 uM). The resulting 3.5‐fold difference in intrinsic clearance for uridine uptake (i.e., Jmax/Km) for hENT1 and hENT2 mediated transport (1.21 mL cm−2 min−1 and 0.35 mL cm−2 min−1, respectively), accounted for the observed inhibition of uridine transport produced by 100 nM NBMPR, indicating that at low concentrations, hENT1 is primarily responsible for uridine uptake in these cells. Together, these data suggest that HeLa S3 cells are an adequate model for studying the characteristics of nucleoside transporters present in the BTB.

Support or Funding Information

Supported by R01GM123643

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multidrug resistance protein 4 (Mrp4) regulates nutrient and energy metabolism by altering the adipose tissue physiology
Authors: Donepudi Ajay C , Lee Yoojin , Lee Ji‐Young , Schuetz John D , Manautou Jose E
Published: June 2021

Multidrug resistance protein 4 (Mrp4; encoded by Abcc4) belongs to the ATP‐binding cassette efflux transporter superfamily and transports several xenobiotics and endogenous molecules, such as prostaglandins and cyclic nucleotides. Mrp4 is highly expressed in the kidney and bladder, with minimal expression detected in the liver. We recently determined that the lack of Mrp4 increases adipose tissue and body weights in mice. Interestingly, the expression of Mrp4 and its role in adipose tissue physiology, nutrient metabolism and the pathogenesis of metabolic diseases are unknown. The current study was aimed at characterizing these specific roles of Mrp4 employing wildtype (WT) and knockout (Mrp4−/−) mice. Our studies determined that Mrp4 is expressed in high levels in adipose tissue and that the lack Mrp4 resulted in adipocyte hypertrophy. Along with increases in body and adipose tissue weights, lack of Mrp4 in mice also resulted in increased blood glucose and leptin levels, and impaired glucose tolerance. Additionally, Mrp4−/− mice have increased levels of adipose tissue phospho‐cAMP response element binding protein (p‐Creb), which is known to regulate adipocyte glucose and lipid metabolism. In contrast to altered glucose metabolism, Mrp4−/− mice did not show any significant changes in plasma triglycerides, free fatty acids or total cholesterol levels compared to WT mice. Indirect calorimetry measurements under basal conditions showed that Mrp4−/− mice have decreased volumetric oxygen consumption (VO2), respiratory exchange ratio (RER), energy expenditure (EE), ambulatory activity (AA) and wheel counts compared to WT mice. Decreased indirect calorimetry parameters and impaired glucose metabolism in Mrp4−/− mice can be attributed to increased adipose tissue weights, which complements the observed obese and diabetic phenotype in these mice. Together, our data indicate that Mrp4 regulates nutrient and energy metabolism by altering the adipose tissue physiology. In conclusion, the absence of Mrp4 function in mice results in the development of obesity and diabetes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Embryonic Stem Cell‐based Vaccine for the Prevention of Lung Cancer
Authors: Meng Shuhan , Whitt Aaron G. , Al Rayyan Numan , Richie Jamaal , Tu Allison , Eaton John W. , Yaddanapudi Kavitha , Li Chi
Published: June 2021

The antigenic similarity between embryos and tumors has raised the idea of using embryonic material as a preventative vaccine against neoplastic disease. Indeed, we have previously reported that a vaccine comprised of allogeneic murine embryonic stem cells (ESCs) and murine fibroblasts expressing immune‐stimulatory granulocyte macrophage‐colony stimulating factor (GM‐CSF) successfully blocks the outgrowth of an implantable lung cancer (Lewis lung carcinoma; LLC) and lung tumors caused by a combination of a mutagen followed by chronic pulmonary inflammation. However, such a vaccine is obviously impractical for application to humans. The use of fibroblasts to produce GM‐CSF is needlessly complicated, and intact live ESCs carry the hazard of generating embryomas/teratomas. Here, we report the successful application of an alternative prophylactic vaccine comprised of exosomes/microvesicles derived from murine ESCs engineered to produce GM‐CSF. The prophylactic potential of the exosomes against implanted (subcutaneous inoculation of LLC) and metastatic (tail vein injection of LLC) lung cancer was tested by immunizing mice with exosomes derived from ESCs. Vaccination of mice with the exosomes bearing GM‐CSF significantly slowed or blocked the outgrowth of LLC while control exosomes lacking GM‐CSF were ineffective. Examination of tumor‐infiltrating immune cells showed robust tumor‐reactive immune responses in mice vaccinated with the exosomes bearing GM‐CSF, including an increase in cytotoxic T cells and a decrease in immunosuppressive T regulatory cells. Overall, vaccination of mice with ESCs‐exo bearing GM‐CSF is very effective in preventing both implantable and metastatic lung tumors with no detectable toxicity. Therefore, we conclude that a similar vaccine derived from GM‐CSF‐expressing human ESCs may be applicable to humans with increased risk of developing lung cancer.

Support or Funding Information

NIH grant CA198249 (K.Y.); Free to Breathe Research Grant (K.Y.); NIH grant AA018016‐01 (J.W.E); Commonwealth of Kentucky Research Challenge Trust Fund (J.W.E.); NIH grants CA106599 and CA175003 (C.L.); 1P30GM106396 Phase III CoBRE grant (C.L.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Excessive salt consumption increases susceptibility to cerebrovascular dysfunction and cognitive impairments in the elderly of both sexes
Authors: Wang Shaoxun , Jiao Feng , Guo Ya , Zhang Huawei , He Xiaochen , Maranon Rodrigo O , Alexandra Barbara , Pabbidi Mallikarjuna , Roman Richard J , Fan Fan
Published: June 2021

Epidemiological study indicates that the prevalence of dementia is higher in women than men. Excessive salt consumption increases the incidence of cardiovascular disorders, which is one of the major risk factors for dementia. However, whether high‐salt intake that can but not always elevate blood pressure contributes to the development of dementia is still not well understood. Moreover, if there is a correlation between excessive salt consumption, cerebral vascular dysfunction, and dementia, especially if there is a relationship between these factors and sex and age is unclear. In the present study, we examined the myogenic response of middle cerebral artery, autoregulation of cerebral blood flow (CBF), functional hyperemia, and cognitive function in young (3‐month) and old (18‐month) SD rats in both males and females. Blood pressure was not altered in all groups of rats fed 8% high salt (HS) diet. Yong and old female rats all displayed more spontaneous tone, less pressure‐induced myogenic response compared to age‐matched males. HS fed rats developed the impaired myogenic response to pressure and to Ach‐induced vasodilation in all age and sex. CBF measured by laser Doppler flowmetry (LDF) rose by 27.7 ± 4.8% (male, young), 57.4 ± 4.4% (female, young), 35.9 ± 3.3% (male, old), and 60.3 ± 12.3% (female, old), respectively, when MAP increases from 100–180 mmHg indicating that female rats have impaired CBF autoregulation in both ages. HS intake diminished and aggravated CBF autoregulation in male and female rats, respectively, in all age. Elderly female rats exhibited impaired functional hyperemia measured by LDF during whisker stimulation compared to age‐matched males. Excessive salt intake also exacerbated the declined functional hyperemia and resulted in cognitive impairment examined by eight‐arm water maze in old male and female rats. These results demonstrated that excessive salt consumption increases susceptibility to the development of cerebrovascular dysfunction and cognitive impairments in both male and female elderly independent of blood pressure. This study provides novel insight to understand the effects of metabolic stressors on sex and age in the development of cerebral vascular function and dementia.

Support or Funding Information

This study was supported by grants AG050049, P20GM104357, DK104184, and HL138685, from the National Institutes of Health; 16GRNT31200036 from the American Heart Association.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Compromised Renal Vasodilations of Adenosinergic Origin in Endotoxic Rats is Reversed by Nicotine: Role of the nAChRs/Heme Oxygenase‐1 Pathway
Authors: Wedn Abdalla M , El‐Gowilly Sahar M , El‐Mas Mahmoud M
Published: June 2021

The nicotinic/cholinergic antiinflammatory pathway protects against acute kidney injury and other end organ damages induced by endotoxemia. In this study we tested the hypothesis that functional α7‐nAChRs/heme oxygnase (HO) pathway is imperative for the nicotine counteraction of renovascular dysfunction caused by endotoxemia in rats. Renal vasodilations were induced by cumulative bolus injections of acetylcholine (ACh, 0.01–7.29 nmol) or ethylcarboxamidoadenosine (NECA, adenosine receptor agonist, 1.6–100 nmol) in isolated phenylephrine‐preconstricted perfused kidneys. The data showed that 6‐hr treatment with i.p. lipopolysaccharide (LPS, 5 mg/kg) elevated biochemical indices of renal function (serum urea and creatinine) and reduced renal vasodilations caused by NECA. The specificity of the LPS interaction with NECA responses was indicated by the lack of similar attenuation in renal vasodilations induced by ACh. Additionally, the LPS effects were not sex‐related because they were observed in both male and female preparations. The attenuated vasodilatory action of NECA and elevated biomarkers of kidney function in endotoxic rats were reversed upon concurrent treatment with bilirubin (5 mg/kg), but not hemin (HO‐1 inducer, 10 mg/kg) or tricarbonyldichlororuthenium (II) dimer (carbon monoxide‐releasing molecule, 10 mg/kg). Likewise, the LPS effects were dose‐dependently abrogated by nicotine co‐treatment (0.5, 1 and 2 mg/kg). The blockade of α7‐nAChRs (methyllycaconitine citrate, 2 mg/kg) or inhibition of HO‐1 (zinc protoporphyrin, 10 mg/kg) blunted the advantageous effects of nicotine on renal endotoxic manifestations. The Kaplan‐Meier survival curve analysis showed that the increase in mortality rate caused by LPS disappeared in the presence of nicotine or bilirubin. Together, the current biochemical and pharmacological evidence suggests key roles for α7‐nAChRs and the bilirubin byproduct of the HO‐1 signaling in the nicotine counteraction of renal dysfunction and reduced adenosinergic vasodilator capacity.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Repeated non‐invasive limb ischemic preconditioning attenuates myocardial ischemia‐reperfusion injury in diabetic rats by up‐regulating hexokinase II
Authors: Zhang Dengwen , Wang Sheng , Ye Xiaodong , Cai Yin , He Yi , Xia Zhengyuan
Published: June 2021

Background

Ischemic heart disease is the leading cause of mortality in patients with diabetes mellitus. Although remote ischemic preconditioning achieved by non‐invasively occluding the brachial artery (non‐invasive limb ischemic preconditioning, NLIP) lost its cardioprotection in diabetes, our recent study demonstrated that repeated NLIP (rNLIP, applying 3 cycles of 5 minutes occlusion/5 minutes reperfusion in a unilateral hindlimb daily for 3 days) reduced myocardial infarction, but the mechanism is unclear. Hexokinase II (HKII) is a key glycolytic enzyme that regulates cell survival when bonds to out mitochondrial membrane. However, it is unknown whether HKII is involved in the cardioprotection of diabetic myocardial ischemia/reperfusion(I/R) injury conferred by rNLIP and warrants investigation.

Methods and Results

Type 1 diabetic rat model was established by intravenous injection of streptozotocin (60 mg/kg). Myocardial I/R was induced by ligating the left anterior descending artery for 30 minutes followed by 2 hours reperfusion. The results showed that HKII was down‐regulated in the myocardium of diabetic rats(p<0.05, vs. non‐diabetic rats), while myocardial I/R injury was aggravated, as evidenced by increases in post‐ischemic myocardial infarct size, lactate dehydrogenase and circulating levels of troponin I (cardiac injury markers, p<0.05 vs. non‐diabetic rats with I/R), concomitantly with enhanced cardiac apoptosis level (reduced Bcl2/Bax ratio, increased cleaved caspase‐3 level and more TUNEL‐staining positive cells) and aggravated inflammatory responses (increased mRNA levels of pro‐inflammatory cytokines IL1β, IL6 and TNFα). rNLIP significantly up‐regulated the expression of HKII in the myocardium of non‐diabetic and diabetic rats (the up‐regulation level is more pronounced in diabetic rats). The rNLIP‐induced HKII expression in diabetic rats was paralleled by reductions in myocardial infarct size, cardiac apoptosis and inflammatory responses.

Conclusion

rNLIP attenuates diabetic heart I/R injury through inhibiting cell apoptosis and inflammatory response via up‐regulation of HKII in cardiomyocytes and that targeting HKII expression may be a potential clinical therapeutic intervention for the management of ischemic heart disease in diabetes.

Support or Funding Information

The study was supported by Guangdong Natural Science Foundation (2018A030313535, Guangdong, China) and HMRF (05161826) of Hong Kong.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aging Healthy, or with Diabetes, is Associated with ACE2/ACE Imbalance in the Hematopoietic Stem Progenitor Cells
Authors: Joshi Shrinidh , Gomez Samuel , Duran‐Mendez Miguel , Quiroz‐Olvera Julio , Garcia Charles , Jarajapu Yagna PR
Published: June 2021

Bone marrow‐derived hematopoietic stem progenitor cells (HSPCs) have vasoreparative functions and are physiologically mobilized into circulation. Aging or diabetes increases risk for cardiovascular diseases and are known to be associated with impaired HSPC functions. Angiotensin converting enzyme (ACE) and ACE2 are primary enzymes of cardiovascular detrimental and protective axes of renin‐angiotensin system (RAS). Angiotensin‐(1‐7) is generated largely by degradation of Ang II by ACE2 and produces vasoprotective functions by activating Mas receptor. We have shown that Ang‐(1‐7) reverses diabetic dysfunction in murine and human HSPCs. This study tested the hypothesis that aging healthy or with diabetes is associated with imbalance in the vascular protective and detrimental axes of RAS in HSPCs. Peripheral blood samples were obtained from either male or female healthy (n=23) or diabetic (n=21), both type 1 and type 2, individuals of age ranging from 25 to 87 years. Lineage‐negative (Lin−) or CD34+ HSPCs were enriched from mononuclear cells. ACE and ACE2 activities in the cell lysates were determined by using enzyme‐specific fluorogenic substrates. Enzyme activities are expressed as arbitrary fluorescence units that is inhibitable by the enzyme‐selective inhibitors of ACE or ACE2, captopril and MLN‐4760, respectively. ACE2/ACE activity ratios in HSPCs in different individuals are plotted against the respective ages. ACE2/ACE is negatively correlated with the age in healthy (Pearson's r=−0.88, P<0.0001) and in diabetic individuals (Pearson's r=−0.82, P<0.0001). As predicted, a negative correlation was observed with ACE2/ACE vs HbA1C (Pearson's r=−0.899, P<0.0001). These results imply that vascular protective axis of RAS is dysfunctional in HSPCs of aging individuals either healthy or diabetic, which is likely to be an underlying mechanism of the impaired reparative functions in HSPCs. Molecular or pharmacological approaches that enhance ACE2 activity or expression could be of great promise in reversing the age‐ or diabetes‐associated dysfunctions and restore cardiovascular health.

Support or Funding Information

National Institute of General Medical Sciences (NIGMS), and National Institute of Aging (AG056881)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ovine Mucosal Enoxaparin Exhibit Comparable Pharmacokinetic Profiles to Porcine Mucosal Enoxaparin
Authors: Rangnekar Varun , Kouta Ahmed , Jeske Walter , Yao Yiming , Hoppensteadt Debra , Niverthi Manoj , Duff Rick , Fareed Jawed
Published: June 2021

Background

Low Molecular Weight Heparins (LMWH) such as Enoxaparin are currently derived from porcine mucosal heparin (PMH), the same depolymerization methods can be used to obtain enoxaparin from unfractionated heparin from other sources. The purpose of this study is to compare the pharmacokinetics of ovine mucosal enoxaparin (OME) with that of porcine mucosal enoxaparin (PME).

Materials and Methods

Branded Enoxaparin (Lovenox, Sanofi‐Aventis, Bridgewater, NJ) and three batches of OME (Suzhou Ronnsi Pharma, Suzhou, China) were used. Following anesthesia, a baseline blood sample was collected from primates (Macaca mulatta; n=4). LMWH was administered subcutaneously (SC) at a dose of 1 mg/kg. Additional blood samples were collected at various times (2,4,6 hours). Anti‐Xa and anti‐thrombin assays were used to calculate pharmacokinetic parameters. Tissue factor pathway inhibitor antigen levels were also measured using an ELISA method.

Results

In the samples collected at 2, 4, and 6 hours, comparable levels of branded PME and the three batches of OME were observed using both anti‐Xa and anti‐thrombin assays. The pharmacokinetic parameters such as biologic half‐life, Cmax, volume of distribution, and clearance were comparable for both the ovine and porcine enoxaparin. Tissue Factor Pathway Inhibitor levels returned to baseline levels by 6 hours in OME, but remained slightly elevated with PME.

Discussion

At a 1 mg/kg SC dosage, three OME preparations produced comparable anti‐Xa and anti‐IIa effects which were similar to the ones observed with PME. The PK parameters calculated based on this data were also comparable for both groups. It is concluded that the SC pharmacokinetics of the three batches of OME were not only comparable between batches, but were also similar to the branded product.

Support or Funding Information

None.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.