bluuebunny/crossref_metadata_2025 huggingface dataset:
BCL6 inhibits CD4+Foxp3+ regulatory T cell trans‐differentiation into macrophage‐like cells via modulation of chromatin long‐range interactions
Authors: Mastascusa Kevin , Lai Bin , Wang Jiwei , Yang William , Sun Yu , Fagenson Alexander , Yu Daohai , Saaoud Fatma , Shao Ying , Cueto Ramon , Wu Susu , Fu Hangfei , Li Honglang , Zhu Peiqian , Wang Hong , Yang Xiaofeng
Published: June 2021

The mechanisms underlying macrophage (Mϕ) heterogeneity and regulatory T cell (Treg) plasticity remain poorly determined. Here, we determined the mRNA expressions of 208 macrophage subset markers, Mϕ transcription factors (TFs) and other regulators in microarray data. We made the following findings: 1) Tissue Mϕ from lung, liver, spleen and intestine (LLSI) express higher M1 markers than adipose tissues (AT); the markers of Mϕ subsets such as MHb, Mhem, and HA‐Mac are AT‐enriched; and the markers of Mox are bone marrow (BM)‐specific; 2) Proadipogenic TFs C/EBPα, PPARγ and proinflamamtory adipokine leptin upregulate M1 markers; 3) Tissue Mϕ from LLSI and BM express higher levels of T cell co‐inhibition receptor CD274 than that of AT, have higher levels of glycolysis, fatty acid pathway, and fatty acid β‐oxidation pathway than that of AT, and prefer using RAB27A, STX3 to RAB31 and YKT6 in mediating exosome biogenesis and docking; 4) The expressions of 31 markers from 10 Mϕ subsets are modulated in eight groups of total 34 diseases; and a wide spectrum of pathological Mϕ pathways are identified from high hierarchical shared pathways to disease‐specific ones; 5) BCL6 inhibits Treg trans‐differentiation into Mϕ‐like cells; 6) BCL6 expression in Treg determines the status of BCL6 high stable Treg and BCL6 low plastic Treg potentially by exosomes generated from tumor cells and other cell types, respectively; and 7) BCL6 deficiency in Treg upregulates Mϕ TFs via increasing the ratio of upstream chromatin long‐range interaction sites (CLRISs) over downstream CLRISs. Our finding identified pathways underlying Mϕ heterogeneity in pathophysiological conditions and Treg plasticity, which serves as novel therapeutic targets for cancers and inflammation.

Support or Funding Information

This work was supported by NIH grant to Drs. XF. Yang, H. Wang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glypican 3 (GPC3) cross talks with HGF/c‐Met axis in hepatocytes and hepatocellular carcinoma (HCC) by modulating p‐Ezrin (Thr567) dependent Yap activity
Authors: Xue Yuhua , Mars Wendy Michelle , Bowen William C , Tao Junyan , Orr Anne V , Stoops John W , Michalopoulos George
Published: June 2021

The experiments that integrate different pathways in normal and abnormal circumstances will help us to develop optimum targeted therapies because of multiple pathways involved in the solid tumor carcinogenesis. We have shown that activation of CD81 is associated with phosphorylation of Ezrin, which is overexpressed in many types of solid tumors and has been identified as metastasis related protein for many years. We have also shown that GPC3 binds to the membrane tetraspanin CD81, which is one of the portals of entry of hepatitis C virus (HCV). Previous studies have shown that GPC3 is associated with inhibition of hepatocyte growth and liver regeneration by inhibiting CD81‐mediated p‐Ezrin/Hippo/Yap pathway. It also has been shown that HGF/Met axis which promotes growth in HCC can phosphorylate Ezrin as well, by different kinases. Since we have demonstrated the predominant GPC3+++/CD81− pattern in HCC, the purpose of this study is to explore the regulation of expression and phosphorylation of Ezrin in normal hepatocytes and HCC. We have found that CD81 expression could be detected in membrane fraction in human hepatoma cell lines HepG2, Huh7 and Hep3B, but immunoprecipitation suggests that CD81 from latter two human hepatoma cell lines has lost association with GPC3. Human hepatoma cell lines have dramatically up regulated Ezrin expression and Yap activity. We found that treatment of primary rat hepatocytes with CD81 agonist antibody or HGF led to higher levels of phosphorylated Ezrin and lower Hippo activity. Mice over‐expressing GPC3 had decreased p‐Ezrin but increased Hippo activity. Rat hepatoma cell lines JM1 and JM2 had dramatically down regulated CD81 expression and Hippo activity, and up regulated p‐Ezrin. In mice with post‐natal Met knock‐out, there was less Yap expression in normal liver. Force expression mutant Ezrin suppressed Hippo activity and activated Yap signaling in vitro and in vivo.

Conclusions

Both HGF/Met and CD81 mediate p‐Ezrin activity and enhance nuclear Yap levels. These pathways in normal hepatocytes are inhibited by GPC3 and low HGF/Met activity. In human hepatocytes, GPC3 inhibits the CD81‐mediated p‐Ezrin effect on Hippo pathway and cause a decrease in nuclear Yap. The different distribution pattern of HGF/Met and GPC3/CD81 between normal hepatocytes and HCC cells provides the chance to develop precise anti‐HCC therapies. And Ezrin is a promising target of anti‐HCC therapies including primary lesion and metastasis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sirtuin 1 (SIRT1) Content and Angiotensin II Receptor Type I (AT1) Expression in Human Umbilical Vein Endothelial Cells (HUVECs) in Response to Resveratrol: Pregnancy Induced Hypertension (PIH) vs Normotensive pregnancy (NTP)
Authors: Szukiewicz Dariusz , Watroba Mateusz
Published: June 2021

Oxidative stress and endothelial dysfunction within uteroplacental unit are inherent components initiating PIH. The normal decrease of vascular angiotensin II sensitivity is lost in PIH. The polyphenol stilbene compound resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene) boosts endothelium‐dependent vasorelaxation enhancing endothelial nitrix oxide synthase (eNOS) production. In fact, resveratrol acts as a secondary regulator influencing more complex biological response that includes the sirtuin system. SIRT1 is highly expressed in endothelial cells and activated/modulated by resveratrol has been shown to target eNOS for deacetylation resulting in enhanced NO‐dependent vascular relaxation. Moreover, anti‐hypertensive effects of resveratrol may be a consequence of the angiotensin II receptor type I (AT1) modulation by SIRT1. The aim of the study was to examine comparatively the effect of resveratrol on SIRT level in HUVECs isolated after PIH vs. NTP. In addition, AT1 expressions were compared. HUVECs were isolated immediately after cesarean section delivery from PIH (N = 24) and NTP (N = 24) patients (group I and group II, respectively). The umbilical cord segments was subjected to collagenase digestion and the HUVECs were cultured in vitro under normoxia. After achieving the confluence, HUVECs were exposed to resveratrol (50, 100 and 250 μM) administered into the culture media in the respective subgroups A, B and C during the 7 day period. SIRT1 levels were determined in HUVECs culture lysates obtained in the days 1,3 and 7, using ELISA. After termination of the cultures AT1 were immunostained in formalin fixed and paraffin embedded sections. To estimate the mean expression of AT1, quantitative immunohostochemistry was applied. The initial AT1 expression was estimated in the respective controls. After 3 and 7 days of the culture, in both groups the mean SIRT1 levels were significantly (p < 0.05) higher in the subgroups B and C, compared to the initial levels at day 1. However, the mean SIRT1 levels in group I were 4.6‐fold and 2.4‐fold lower (the subgroups B and C, respectively) than observed in normotensive group II. The initial expression of AT1 was significantly higher in group I and amounted to 170% of the average value in the normotensive group II. The mean expression of AT1 was significantly (p < 0.05) downregulated in group I (the subgroups B and C), while in group II was nearly unaffected. These results may suggest potential beneficial effect of SIRT1 activation by resveratrol with regard to antihypertensive therapy in PIH.

Support or Funding Information

WUM grant: 2M2‐W1‐17

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of the Effect of Phototherapy on the Inflammatory Mediators in an Experimental Model of Ischemic Vascular Accident
Authors: Albertini Regiane , Vogel Debora , Villatoro Nancy , Araújo Noemi , Marques Marcia , Scorza Carla , Aimbire Flavio , Scorza Fulvio , Cavalheiro Esper
Published: June 2021

Cerebral Vascular Accident (CVA) is a sudden neurological condition, which mostly results from cerebral ischemia. Responsible for a large number of deaths and functional disability worldwide, it is extremely important to implement preventive strategies and identify new therapies. Phototherapy has been an excellent therapeutic modality for several diseases, few studies have explored this therapeutic modality for the treatment of stroke. The aim of this study was to analyze whether the treatment with LLLT is able to modulate the inflammatory mediators in the experimental model of ischemic stroke (AVCI). The study was approved by the Ethics Committee on Animal Use of the Federal University of São Paulo (CEUA / UNIFESP) under the number 3316110516. The investigation was conducted in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education. The AVC was induced by the photothrombosis method in 8 male Wistar rats (230 ± 20g) randomly assigned to two experimental groups: Group AVC Control (AVCC) and Group AVC Laserterapia (AVCL). The application of transcranial LLLT was done using a laser diode (MM Optics), with wavelength of 780 ± 5nm, power density of 10 mW / cm2, for 2 minutes, 3× / week for a period of 2 months . The animals were euthanized with anesthetic overdoses of Barbituric / Thiopental Sodium (Cristália) at a dose of 80 mg / kg intraperitoneally; transcardiac perfusion was performed and then the biological tissue (brain) was extracted. The tissues were frozen in liquid nitrogen. The levels of TNF‐α, IL‐1β, IL‐6, IL‐10, VEGFA, BDNF, IGF1, TGFB1 and ANGIOTENSIN II in the samples were determined by the enzyme‐linked immunosorbent assay (ELISA), following commercial kit instructions (R & D System, USA). ). Data were analyzed by the Shapiro‐Wilk normality test in order to verify that the samples come from populations with normal distribution; then the variance test was performed using the Snedecor F test, and then the unpaired T‐test was used; for all tests was considered α = 5%. Our results indicate that there was modulation of the IL1‐b mediators (AVCC 0.4075 ± 0.156659 × AVCL 0.232091 ± 0.083417 p <0.01); IL‐6 (AVCC 180.5753 ± 49.70076 × AVCL 25.47956 ± 15.58209; p <0.001); TNF (40.06277 ± 17.23243 × AVCL 18.2546 ± 5.8065; p <0.01), and in relation to IL‐10 and TGF‐β there were no significant changes. We can conclude that phototherapy modulates the inflammatory response , This result may indicate that the area of shadow of the lesion could be smaller favoring recovery after AVC.

Support or Funding Information

Financial Support: Foundation for Research Support of the State of São Paulo (FAPESP) 2016 / 10286‐7

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increasing upstream chromatin long‐range interactions may favor induction of circular RNAs in lysoPC‐activated human aortic endothelial cells
Authors: Li Angus , Sun Yu , Drummer Charles , Yu Daohai , Zhou Yan , Li Xinyuan , Johnson Candice , Yu Caijia , Yang William , Jiang Xiaohua , Hu Wenhui , Wang Hong , Yang Xiaofeng
Published: June 2021

Circular RNAs (circRNAs) are classified as non‐coding RNA that form covalently closed continuous loops and act as gene regulators in physiological and disease conditions. We hypothesize that proatherogenic lipid lysophosphatidylcholine (LPC) induce a set of circRNAs in human aortic endothelial cell (HAEC) activation. We performed circRNA analysis by searching our RNA‐Seq data from LPC‐activated HAECs and found: 1) LPC induce significant modulation of 77 newly characterized cirRNAs, among which 47 circRNAs (61%) are upregulated; 2) 34 (72%) out of 47 upregulated circRNAs are upregulated when the corresponding mRNAs are downregulated, suggesting that the majority of circRNAs are upregulated presumably via LPC‐induced “abnormal splicing” when the canonical splicing for generation of corresponding mRNAs is suppressed; 3) Upregulation of 47 circRNAs is associated with LPC‐upregulated cholesterol synthesis‐SREBP2 pathway and LPC‐downregulated TGF‐β pathway; 4) increased upstream chromatin long‐range interaction sites to circRNA related genes favor circRNA generation over canonical splicing for mRNAs, suggesting that shifting chromatin long‐range interaction sites from downstream to upstream promotes induction of a list of circular RNAs in lysoPC‐activated human aortic endothelial cells; 5) six significantly changed circRNAs may have sponge functions for miRNAs; and 6) 74% significantly changed circRNAs contain open reading frames, suggesting that short proteins may interfere the protein interaction‐based signaling. Our finding have demonstrated for the first time that a new set of LPC‐induced circRNAs may contribute to proatherogenic LPC‐induced HAEC activation. These novel insights may lead to identification of new therapeutic targets for treating metabolic cardiovascular diseases, inflammations and cancers.

Support or Funding Information

This work was supported by NIH grants to Drs. XF Yang and H Wang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Active Learning in a Pharmacology Classroom
Authors: Ayanniyi Rashidat Oluwafunke , Olumoh‐Abdul Hidayah Ayodeji
Published: June 2021

Pharmacology teaching in Nigeria is extremely teacher‐centered and does not meet the educational needs of Pharmacy students. Despite the numerous advantages of active learning, Pharmacy schools in Nigeria are yet to adopt this teaching method.

This action research was carried out to introduce and determine the effectiveness of active learning in a pharmacology classroom. Twenty‐four Pharmacy students in their third year participated in the study. Randomized control‐group pre‐test post‐test design was used. A pre‐test was administered to the control and treatment groups. Traditional lecture method was employed in teaching twelve students (control group) while three active learning strategies; Think‐pair‐share, Student presentation and Socratic questioning was used in teaching a cardiovascular module to twelve students (treatment group). A Post‐test was given to both groups after four weeks of instruction. We administered a structured questionnaire to obtain information on students' perception of active learning strategies. A higher mean score was obtained from the post‐test of students taught with active learning strategies. Majority of the respondents agreed active learning enhanced team work, increased their knowledge and confidence. Inadequate pre‐class preparation by the students was a major barrier encountered with use of active learning strategies and more time will be required to depart from passive to active learning. In conclusion, students were involved in the learning process and effective instruction was achieved. These findings will be of value to educators who plan to adopt active learning strategies in small classrooms.

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.
Therapeutic Potential of G‐protein‐Biased Kratom‐Derived and Synthetic Carfentanil‐Amide Opioids for Alcohol Use Disorder
Authors: Gutridge Anna M , Robins Meridith T , Cassell Robert J , Uprety Rajendra , Mores Kendall L , Ko Mee Jung , Pasternak Gavril W , Majumdar Susruta , van Rijn Richard M
Published: June 2021

Mitragyna speciosa, also known as kratom, is a plant containing opioidergic alkaloids and is currently being used to self‐medicate chronic pain, opioid dependence, and is also used as a heroin substitute. Kratom, though unscheduled, may be less dangerous when compared to prescription opioids due to the preferential interaction of kratom alkaloids with G‐proteins instead of beta‐arrestin proteins. We hypothesized that kratom alkaloids and synthetic opioids with similar biased pharmacology can be used to treat alcohol use disorder. We completed in vitro pharmacological characterization for two kratom strain extracts, four kratom alkaloids (mitragynine, 7‐hydroxymitragynine, paynantheine, and speciogynine), and a series of synthetic carefentanil‐amide opioids by determining their ability to interact selectively with G‐proteins instead of beta‐arrestin at the mu, delta and kappa opioid receptors. In addition, in C57BL/6 mice we assessed if kratom (alkaloids) and opioids were capable of reducing alcohol use and determined if the alkaloids had rewarding properties. We found that kratom alkaloids are strongly biased towards the G‐protein pathway at all three opioid receptors. More specifically, we found the delta opioid receptor plays an important role in the mechanism of action as interpreted from reduced efficacy in knockout mice. MP102, one of the synthesized carfentanilamide opioids, displayed a pharmacological profile similar to the kratom alkaloids but with preference for delta opioid receptors, and also reduced alcohol intake and likewise produced less significant place preference than morphine. Our findings demonstrate that kratom use can decrease alcohol use, but its use still carries substantial risk. The development of further delta‐opioid‐selective synthetic opioids may provide an avenue for alcohol use treatment that does not produce strong rewards.

Support or Funding Information

RMvR support: NIH grants AA025368, AA026949, AA026675, DA045897, a NARSAD young investigator award from The Brain and Behavior Research Foundation, the Purdue Institute for Drug Discovery and the Dept. of Med.Chem. and Molecular Pharmacology. SM support: NIH grants DA045884, DA046487, AA026949 and W81XWH‐17‐1‐0256 & start‐up funds from the Center for Clinical Pharmacology in St. Louis. Funded in part through NIH/NCI Cancer Center Support Grant P30. CA008748.

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 Decline of Glutamate‐Stimulated Norepinephrine Release in Aging Rat Brain
Authors: Dezfuli Ghazaul , Martin Lukas , Olson Thao , Kellar Kenneth J.
Published: June 2021

Unravelling and understanding the molecular and neurochemical changes of the aging brain is key to early intervention strategies. Glutamate (Glu) neurotransmission, the major excitatory neurotransmitter in the brain, is mechanistically involved in memory, learning performance, and other aspects of cognition. Moreover, as the brain ages, the glutamatergic signaling system mediated through brain N‐methyl‐D‐aspartate (NMDA) receptors becomes hypofunctional, and this decline may be associated with cognitive dysfunction in normal aging and in disease states. We have developed an in vitro assay that measures Glu‐stimulated release of neurotransmitters in rat brain slices. Using this assay, we compared changes in Glu‐stimulated release of [3H]‐norepinephrine (NE) from the cerebral cortex and hippocampus in young (2–3 months old) versus old (21–23 months old) Fischer F344 rats. Our findings demonstrate that that there is a pronounced deficit (approximately 30–40%) of Glu‐stimulated [3H]‐NE release in the aged rat brain in comparison to younger controls. In the same tissues, potassium (K+) stimulated‐NE release is similar in brains from young and old rats, suggesting that the age‐related decline in in Glu‐stimulated NE release is related to a change in NMDA receptors. Notably, the deficit in Glu‐stimulated [3H]‐NE release in both brain regions was completely rescued by the addition of 1 uM d‐amphetamine sulfate to the tissue slices. Overall, these findings reveal an important deficit in NE release in the aging brain, which may have important implications for cognitive dysfunction in aging. Moreover, this deficit can be reversed with amphetamine and possibly with similar drugs. These studies have clear clinical implications for the use of CNS stimulants as a therapeutic intervention for early cognitive impairment.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Treatment with polyamine oxidase inhibitor reduced neurodegeneration and improved retinal function in diabetic mice
Authors: Liu Fang , Saul Alan B , Xu Zhimin , Palani Chithra D , Shosha Esraa , Caldwell Ruth B , Narayanan S. Priya
Published: June 2021

Purpose

Deregulation of polyamine metabolism has been implicated in various neurodegenerative disease and injury conditions. Studies from our laboratory have demonstrated that polyamine oxidation is a critical mediator of both neuronal and vascular injury in a mouse model of retinopathy of prematurity (Narayanan et al 2014; Patel et al 2016). The current study was undertaken to investigate the role of the polyamine oxidase spermine oxidase (SMO) in mediating neuronal damage and dysfunction in the diabetic retina.

Methods

Mice (8 weeks old, C57BL6J) were made diabetic by injection of streptozotocin (STZ, 65 mg/kg, pH 4.5, i.p.) and compared to age‐matched controls after 4 to 15 weeks of diabetes. Following the onset of hyperglycemia, mice were treated with either vehicle (normal saline) or SMO inhibitor, MDL 72527 (20 mg/kg of body weight, in saline, intra‐peritoneal, 3 times/week). Retinal neuronal function in living mice was determined by electroretinography (ERG). Retinal structure in the same mice was analyzed by Spectral Domain‐Optical Coherence Tomography (SD‐OCT). Following ERG and SD‐OCT analyses, mice were humanely euthanized, and retinas were collected and processed for immunofluorescence and western blot analyses.

Results

Western blot analysis showed that expression of SMO was increased in the diabetic retinas compared to controls. Inner retinal function studied by dark‐adapted electroretinography showed that the positive (pSTR) and negative (nSTR) scotopic threshold responses (measured at 110 ms and 200 ms), were significantly reduced (p<0.01, N=5–12) in the diabetic (4–12 weeks) mice as compared with the controls. The pSTR, but not the nSTR amplitudes were greatly improved in the MDL‐treated diabetic mice (p<0.05, N=5–10). The amplitude at 110 ms was dominated by the b‐wave at higher intensities and was significantly stronger in the MDL72527‐treated mice than in the vehicle‐treated diabetic mice. SD‐OCT analysis demonstrated a significant thinning of retina in the diabetic mice (p<0.01, N=5–12), which was improved in the MDL‐treated group. These data demonstrate a partial rescue of inner retinal function and retinal structure by SMO inhibition in the diabetic retina. Studies on the impact of MDL72527 treatment on neuronal survival are currently in progress.

Conclusion

Our results show the specific involvement of Spermine Oxidase in mediating neuronal injury in the diabetic retina and demonstrate its potential as a therapeutic target for mitigating neuronal injury in diabetic retinopathy.

Support or Funding Information

These studies supported by the National Eye Institute (1R01EY028569 to S.P.N) and Augusta University Culver Vision Discovery institute pilot grant to S.P.N.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ca2+ allostery in PTH‐receptor signaling
Authors: White Alex , Sutkeviciute Ieva , Jean‐Alphonse Frederic , Xiao Kunhong , Vilardaga Jean‐Pierre
Published: June 2021

The parathyroid hormone (PTH) type 1 receptor (PTHR) serves as the cognate receptor for two endogenous ligands, PTH and its related peptide (PTHrP). Binding of PTHrP to the PTHR results in only transient cAMP production that is limited to the plasma membrane, while PTH induces sustained cAMP responses derived from endosomes following internalization of the ligand‐receptor complex. The underlying mechanism and regulation of these kinetically‐distinct signaling events are poorly understood. Here we show that extracellular Ca2+ acts as a positive allosteric modulator of the PTHR that promotes sustained cAMP production. Equilibrium and kinetic analysis of ligand binding and receptor activation revealed that extracellular Ca2+ increases the residence time of ligands on the receptor, consequently increasing the duration of receptor activation and cAMP production. Additionally, we found that this sensitivity to Ca2+ is lost for the PTH mutant R25C, identified as a cause of hypocalcemia in human patients. Using mass spectrometry (MS) approaches, we identified acidic clusters in the first extracellular loop of PTHR that serve a critical role in mediating Ca2+ allostery and sustained cAMP signaling. These findings provide insights toward understanding the determinants of PTHR‐mediated signaling events and how these processes are regulated.

Support or Funding Information

Nothing to Disclose: AW, IS, FGJ, KX, JPVSources of Research Support: the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) and the National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH) under grant Awards Number R01‐DK102495, R01‐DK111427, and R01‐DK116780 (to J.P.V.), and T32‐GM008424 (to A.D.W.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Negative Allosteric Modulation of Arrestin Recruitment to the β2‐Adrenergic Receptor
Authors: Ippolito Michael , Hopfinger Nathan , Nayak Ajay P. , Sakkal Leon A. , Rajkowski Kyle Z. , Liu Xiangyu , Kobilka Brian K. , Penn Raymond B. , Armen Roger S. , Scott Charles P. , Benovic Jeffrey L.
Published: June 2021

The β2‐adrenergic receptor (β2AR) is a Gs‐coupled receptor that promotes an agonist‐dependent cyclic AMP (cAMP)‐mediated reversal of bronchoconstriction that is therapeutic for airway diseases like asthma. Long‐term use of β‐agonists is associated with arrestin mediated desensitization of response to treatment. Gs‐biased signaling through the β2AR would have clinical utility in promoting airway relaxation while avoiding desensitization of response to β‐agonists. Using bioluminescence resonance energy transfer (BRET) for arrestin recruitment, cAMP ELISAs, radioligand assays, mutagenesis, X‐ray crystallography, and ex vivo airway tissue models, we characterized compounds that inhibit arrestin recruitment to the β2AR without inhibiting cAMP production in the presence of β‐agonists. Our data reveal a druggable allosteric site in the extracellular vestibule of the β2AR that can be targeted to modulate arrestin recruitment to the receptor without affecting Gs coupling. Here we characterize a small molecule that targets this site to promote β2AR specific, negative allosteric modulation of arrestin recruitment that is protective against agonist‐induced desensitization in cells and airway tissue.

Support or Funding Information

Research funded by NIH training grant GM100836, P01 HL114471, and F31 HL139104.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Pharmacology and Structure–Activity Relationships of D1 Dopamine Receptor Positive Allosteric Modulators
Authors: Luderman Kathryn D. , Free R. Benjamin , Aubé Jeffrey , Frankowski Kevin J. , Sibley David R.
Published: June 2021

The D1 dopamine receptor (D1R) is a key component of regulating dopaminergic signaling and, as such, is associated with many important neurological processes and diseases. It is an attractive target to treat a wide range of neuropsychiatric disorders, such as Parkinson's disease and the cognitive decline associated with schizophrenia and Alzheimer's disease. Positive allosteric modulators (PAMs) of the D1R have emerged as a drug development strategy, as D1R orthosteric agonists have clinical liabilities curtailing their use. We discovered two D1R PAMs via a high‐throughput screen, MLS1082 and MLS6585. The two PAMs act similarly, potentiating agonist‐stimulated G‐protein and β‐arrestin‐mediated signaling and increasing dopamine's affinity for the D1R. Neither compound has any inherent agonist activity in the absence of dopamine. MLS1082 and MLS6585 are structurally distinct chemical series and we have found they act at two separate binding sites. MLS1082 shares a similar binding site with two known D1 PAMs, Compound B and DETQ, which involves the R130 amino acid in the second intracellular loop of the D1R. MLS6585 displays additive activity with MLS1082, Compound B, and DETQ and its activity is not affected by a R130Q point mutation, strongly suggesting that MLS6585 acts at a separate binding site than these other D1R PAMs. Though D1R PAMs have been described by our group and others, the pharmacology and structure–activity relationships (SAR) underlying D1R allosteric modulation is not well understood. We used several analog sets of both MLS1082 and MLS6585 in order to begin addressing these questions as well as to improve on the D1R PAM activity of the parent compounds. Many of the analogs of both MLS1082 and MLS6585 retained PAM activity, validating these drug scaffolds as D1R modulators. The analogs of one series displayed additive activity with the opposite series, suggesting that the analogs are binding to similar sites as their parent compounds. The largest improvement on the parent compounds came via better selectivity for the D1R over the closely related D5 dopamine receptor. This has important implications for understanding the homology of allosteric sites between related receptor types. A small number of seemingly inactive analogs appear to act as silent allosteric modulators (SAMs), in that they blocked the activity of the parent PAM compound, presumably by competing for the same binding site. Further analog studies are underway to better understand the chemical SAR needed for D1R allosteric modulation as well as the differences between the two D1R PAM binding sites.

Support or Funding Information

Funded by NINDS Intramural Research Program and Eshelman Institute for Innovation

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 Family of Photoswitchable Metabotropic Glutamate Receptors for High‐Efficiency Optical Interrogation of Specific Receptor Populations in vivo
Authors: Gutzeit Vanessa , Ruiz Amanda Acosta , Broichhagen Johannes , Levitz Joshua
Published: June 2021

G protein‐coupled receptors (GPCRs) form a large family of membrane bound receptors that initiate intracellular responses which control cellular signaling cascades to contribute to a wide range of biological functions. The metabotropic glutamate receptors (mGluRs) consist of an eight‐member family of class C GPCRs that respond to the excitatory neurotransmitter glutamate and serve as promising pharmacological targets for a range of neurological and psychiatric disorders. mGluRs are localized in a variety of cell types throughout the central nervous system where they can modulate neuronal activity in both normal and disease‐related states. Despite the prevalence of these receptors, the underlying molecular, synaptic and circuit mechanisms of mGluR‐mediated neuromodulation are still poorly understood due to the limitations of classical pharmacological and genetic approaches. Here we report an improved chemical optogenetic toolkit that permits the reversible and repeatable activation of specific mGluRs with genetic targeting, subtype‐specificity and spatiotemporal precision. Using an updated family of azobenzene‐based photoswitchable glutamate compounds that covalently attach to protein tags, we have dramatically enhanced the efficiency and applicability of these tools. A branched SNAP‐targeting compound termed “doubleBGAG” enables near‐complete photoagonism of mGluR2 when compared to saturating glutamate. The improved efficiency of SNAP‐mGluR2 activation by doubleBGAG permits superior optical inhibition of cortical neurons. Strikingly, doubleBGAG also drastically improves the efficiency of photoagonism of SNAP‐mGluR3 from <10% to ~85% compared to saturating glutamate, allowing investigation of group II mGluR signaling with enhanced subtype specificity. In addition, development of a chimera‐based approach allows for photoactivation of mGluR5 signaling with sub‐cellular targeting. Branched photoswitches targeting CLIP or Halo‐tags along with spectral variants allow multiplexing optical control of multiple receptors. Toward the ultimate goal of targeted delivery of photoswitches to native mGluRs, doubleBGAG also enhances photoswitch efficiency when conjugated to GFP‐targeting nanobodies. Finally, we report the development and testing of fluorophore‐conjugated photoswitches, which allow dual photoactivation and detection of mGluR2 in rodent medial prefrontal cortex in acute slice preparations and in vivo. Together this optogenetic toolkit allows for the dissection of mGluR subtype‐specific contributions to physiological and behavioral changes in mouse models of psychiatric disorders.

Support or Funding Information

NIH 2018 R35 GM: 1R35GM124731‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CP‐25 suppressed the synovial pannus formation in experimental arthritis through Angiotensin □ and its signals
Authors: Yan Shangxue , Cheng Zhuoyu , Wei Wei
Published: June 2021

Object

Rheumatoid arthritis (RA) is a chronic systemic, autoimmune and inflammatory disease represented as synovitis, pannus formation, adjacent bone erosions, and joint destruction. Paeoniflorin‐6′‐O‐benzene sulfonate (code: CP‐25), a novel ester derivative of paeoniflorin, was evaluated the effects of anti‐angiopoiesis in rats with collagen‐induced arthritis (CIA) and regulated Angiotensin □(Ang□) and its signals pathway on vein endothelial cells.

Methods

CIA rats were treated with CP‐25 (50 mg/kg) from days 14 to 35 after immunization. The joint synovial hemodynamics of rats were detected by ultrasonographic methodology and the synovial pannus formation was observed by histopathologic assay. To detect the role of CP‐25 on the synovial pannus formation, human umbilical vein endothelial cells (HUVECs) were co‐stimulated with Ang □ (10−7 mol/L) and TNF‐α (5 ng/ml) in vitro, and CP‐25(10−5 mol/L), Losartan (10−5 mol/L, AT1R blocker) or PD123319 (10−5 mol/L, AT2R blocker) were individual or combined treatment. The effects of CP‐25 on HUVECs functions such as proliferation, migration were detected by Cell Counting Kit‐8 assay (CCK8) or transwell assay. The tube formation were examined by using Matrigel model. The protein expression of AT1R/AT2R signaling pathways in HUVECs were analyzed by western blot.

Results

CP‐25 could alleviate pathological changes and inhibit the synovial pannus formation in CIA rats, decrease regional synovial blood flow signals in the local swelling knee joint. CP‐25 could inhibit HUVEC proliferation, migration as well as tube formation which co‐stimulated with Ang□ and TNF‐α in vitro. CP‐25 showed a synergistic role with Lostan (AT1R antagonist) on down‐regulated the expression of GRK2, p‐ERK1/2, p‐IκBα and p‐P65. Meanwhile, CP‐25 could also inhibit the high level of GRK2, p‐ERK1/2, p‐IκBα and p‐P65 which induced by AT2R antagonist (PD123319).

Conclusion

CP‐25 could present a potent anti‐angiopoiesis effect in CIA rats and inhibit proliferation, migration as well as tube formation function of the HUVEC through Ang□ and its signals mediating AT1R/AT2R‐GRK2‐ERK1/2‐NF‐κB pathway.

Support or Funding Information

This research was supported by the National Natural Science Foundation of China (No. 81330081; 81673444)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of NLRP3‐Specific Inflammasome Inhibition on Ventricular Arrhythmias in Post‐Infarcted Rats
Authors: Lee Tsung‐Ming , Lin Shinn‐Zong
Published: June 2021

Myocardial infarction activates the NLRP3 inflammasome that triggers the inflammatory caspase‐1 activation and the IL‐1β maturation. We studied whether a specific inhibitor of the NLRP3 inflammasome, CP‐456,773, can attenuate cardiac sympathetic reinnervation and fatal arrhythmias in a rat model of myocardial infarction. Male Wistar rats were subjected to coronary ligation and then randomized to either saline or CP‐456,773 in in vivo and ex vivo studies. Postinfarction was associated with the activation of NLRP3 inflammasome components and increased the protein and expression of IL‐1β. Immunohistochemical staining of the border zone after infarction revealed macrophages positive for IL‐1β at 3 days post‐MI. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle‐treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with CP‐456,773 significantly attenuated myocardial levels of mature caspase‐1 and IL‐1β proteins although there were similar levels of NLRP3 and ASC proteins. CP‐456,773 did not affect NLRP3, ASC, caspase‐1 and IL‐1β expression at mRNA levels. Sympathetic hyperinnervation was blunted after administering CP‐456,773, as assessed by immunofluorescent analysis and western blotting and real‐time quantitative RT–PCR of nerve growth factor. Arrhythmic scores in the CP‐456,773‐treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed no additional effect of anti‐IL‐1β antibody on attenuated levels of NGF compared with CP‐456,773 alone. CP‐456,773 protects ventricular arrhythmias by attenuating sympathetic innervation via NLRP3 inflammasome/IL‐1β‐dependent pathways, which converge through the attenuated formation of nerve growth factor in infarcted rats.

Support or Funding Information

nil

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuropilin‐1 Deficient Macrophages as a Treatment Strategy for Glioblastoma
Authors: Caponegro Michael D. , Tsirka Stella E.
Published: June 2021

Post‐diagnosis survival of grade IV glioblastoma (GB) is a dismal 14 months in patients receiving standard of care therapy. With the development of immunotherapeutic strategies targeting non‐cancerous, stromal cells of the tumor microenvironment (TME), new avenues are being pursued in clinical trials and pre‐clinical animal models of advanced GB. Glioma‐associated microglia/macrophages (GAMs) of the TME promote chemoattraction, immune suppression, neoangiogenesis, and tumor cell survival, which parallels tumor‐infiltrating macrophage behavior of other solid malignancies. GAMs are now recognized as a potentially practical immunotherapeutic target in GB. Previously in our lab, we have shown that GAM‐specific deletion of the cell surface co‐receptor Neuropilin‐1 (NRP1) drastically reduces pro‐tumorigenic behavior of infiltrating microglia and/or macrophages in murine glioma by reducing immunosuppressive tendencies and neoangiogenesis. As an extension to our genetically‐ablated mouse model, we present here a platform by which these NRP1‐deficient monocytic cells can be harvested ex vivo and locally delivered into the TME of the bulk glioma lesion, to exert anti‐tumorigenic effects. To our knowledge, this is the first attempt to deliver cells of monocytic origin intratumorally as a therapeutic option.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nicotinic Acetylcholine Receptors of α7 and α4β2 Types Mediate the Nicotine Counteraction of Impaired Baroreceptor Function in Endotoxic Rats
Authors: Sallam Marwa Y , El‐Mas Mahmoud M , El‐Gowilly Sahar M
Published: June 2021

Autonomic dysfunction and reduced arterial baroreceptor activity are prominent features of sepsis. Given the defensive role of the cholinergic antiinflammatory pathway against endotoxic manifestations, agonist and antagonist studies were employed in the current study to identify the roles of α7 and α4β2 nAChRs in impaired reflex chronotropic function caused by endotoxemia in conscious male rats. Endotoxemia was induced by i.v. administration of lipopolysaccharides (LPS, 10 mg/kg) and baroreflex activity was measured by the vasoactive method. The latter involves the assessment of reflex chronotropic responses evoked by increases (phenylephrine, PE) or decreases (sodium nitroprusside, SNP) in blood pressure. Slopes of baroreflex curves were taken as a measure of baroreflex sensitivity (BRS). The data showed that the shifts caused by LPS in baroreflex curves generated by PE or SNP as well as the associated decreases in slopes of the curves (BRSPE, from −2.0±0.2 to −1.2±0.1 beats/min/mmHg; BRSSNP, from −3.7±0.3 to −2.1±0.2 beats/min/mmHg) were dose‐dependently reversed by subsequent i.v. administration of nicotine (25, 50, or 100 μg/kg). Such nicotine effects disappeared after selective blockade of α7‐nAChRs or α4β2‐nAChRs by intracisternal administration of methyllycaconitine (MLA) and dihydro‐β‐erythroidine (DHβE), respectively. We also report that the corrective effects of nicotine on BRSPE were replicated in rats treated with i.v. PHA‐543613 (α7‐nAChR agonist; 0.2–2.0 mg/kg) or 5‐iodo‐A‐85380 (5IA, α4β2‐nAChRs agonist; 0.01–0.1 mg/kg) in dose‐related fashions. Conversely, while the depressed BRSSNP of endotoxic rats was preserved after individual treatment with PHA or 5IA, improved BRSSNP was only seen after the combined exposure to the two drugs. Collectively, functional α7 or α4β2 nAChRs is essential for the demonstration of the alleviation by nicotine of baroreflex dysfunction induced by endotoxemia. Moreover, the precise roles of the homomeric α7 and heteromeric α4β2 nAChRs depend on the nature of the reflex chronotropic response (bradycardia vs. tachycardia).

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.
Involvement of KCa2.3 Channels in Relaxation of Erectile Tissue is Altered in Type 2 Diabetic Mice
Authors: Steffensen Simon Gabriel Comerma , Fais Rafael Sobrano , Beck Lilliana , Mogensen Susie , Simonsen Ulf
Published: June 2021

Introduction

Activation of endothelial small conductance calcium‐activated K+ channels (KCa2.3 or SK3) and intermediate conductance calcium‐activated K+ channels (IK or KCa3.1) leads to vascular relaxation. Our previous studies have shown that KCa2.3 down‐regulation diminishes erectile function; this channel it is the most expressed KCa channel in the endothelium of corpus cavernosum. In the present study we hypothesized that KCa2.3 channel function is altered in erectile tissue of type 2 diabetic animals.

Materials and Methods

Erectile function was measured in diabetic db/db mice and was compared to heterozygous control animals. Corpus cavernosum strips were mounted for isometric tension recording, and they were processed for qPCR or Western Blot. The current research was performed in accordance to FASEB Statement of Principles for the use of Animal in Research and Education.

Results

In anesthetized diabetic db/db mice erectile function was diminished compared to control animals. Concentration‐dependent contractions to noradrenaline were increased in corpus cavernosum strips from db/db compared to strips from control mice. Apamin, a blocker of KCa2 channels inhibited acetylcholine relaxation in corpus cavernosum from db/db and control animals, being less effective in db/db compared to control animals. NS309 (0.5 μM), an activator of KCa2 and KCa3.1 channels, potentiated concentration‐response curves for acetylcholine in corpus cavernosum from control, but this was not the case in corpus cavernosum from db/db mice. SNP relaxation was similar in corpus cavernosum from db/db and control animals. KCa2.3 gene and protein expression was increased in corpus cavernosum from db/db mice, nevertheless protein expressed in aorta was unchanged.

Conclusions

Our results suggest that in type 2 diabetes KCa2.3 channel expression is unchanged in the systemic circulation, and upregulated in erectile tissue. In erectile tissue noradrenaline contraction is increased and the effect of NS309 reduced which may be related to impaired KCa2.3 channel function. These changes may contribute to impairment of erectile function in diabetes.

Support or Funding Information

Aarhus University, Novo Nordisk Foundation and the Danish Research Council.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glucagon receptor antagonists for diabetes mellitus treatment: A systematic literature review
Authors: Miyama Kelle , Elbarbry Fawzy , Bzowyckyj Andrew
Published: June 2021

Objective

The objective of the study was to evaluate the available literature on the safety and efficacy of glucagon receptor antagonists for the treatment of diabetes mellitus.

Methods

A search of PubMed using the terms “glucagon receptor antagonist AND diabetes” and “Pharmacokinetics AND pharmacodynamics AND glucagon receptor antagonist” was conducted. Initial study selection identified 398 articles. Titles and abstracts were reviewed for relevance, resulting in a total of 377 articles excluded (e.g. use of GLP‐1 or GIP agonist, combination therapy). The full texts of the remaining articles were evaluated and their references reviewed to identify additional articles meeting inclusion/exclusion criteria (n=2); resulting in 23 total studies included.

Results

Of the current literature evaluated, 13 studies were conducted in animal models, whereas 11 studies were conducted in humans, with one study occurring in both. A few compounds were identified as having promise for diabetes management including: LY2409021, LGD‐6972, REGN1193, Bay 27‐9955, PF‐06291874, MK‐3577. Findings from the animal and human studies were similar. Both study types demonstrated consistent glucose lowering effects with low rates of hypoglycemia, but varied by agent in regards to alterations in other surrogate measures (e.g. blood pressure, HDL‐C, LDL‐C, Triglycerides, AST, ALT), pharmacokinetics, and adverse effect patterns. Most adverse effects identified were minor and reversible. Efficacy of glucose lowering effects in animal models showed a reduction in blood glucose ranging in magnitude of 57–98 mg/dL. Human models demonstrated a lowering in FPG ranging in magnitude of 27–57 mg/dL.

Conclusion

Glucagon receptor antagonists have shown promise in animal and human models as an alternative option for diabetes mellitus treatment. The glucose lowering benefit with low rates of hypoglycemia is promising. Ongoing research is needed to further assess their efficacy and safety over the long‐term.

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