bluuebunny/crossref_metadata_2025 huggingface dataset:
A Drosophila genetic screen for the discovery of novel NF‐kB and apoptotic regulatory genes
Authors: Jones Rheinallt , Luo Liping , Neish Andrew
Published: June 2021

The Aeromonas protein AopP is a member of the YopJ‐like family of effectors which are secreted by certain pathogens during host invasion. AopP is known to modulate potent immunosuppressive and pro‐apoptotic effects in mammalian cells via inhibition of the NF‐kB pathway at the level of IKK. We have studied the effects of AopP in Drosophila, a model system with functional conservation between its innate immune signaling pathways and those of mammals. We created transgenic Drosophila harboring aopP, allowing directed expression of AopP in Drosophila tissues. AopP expression in immune challenged flies inhibited upregulation of NF‐kB responsive genes, while mutant AopP did not. Interestingly, AopP expression in the Drosophila retina resulted in an altered eye phenotype characteristic of elevated levels of apoptosis during development. We have used this altered eye phenotype as a scorable marker in an EP‐element based genetic screen. EP‐element lines that rescue the AopP apoptotic eye phenotype are considered to have a putative genetic interaction with AopP. This technique allows recovery of previously uncharacterized genes whose identities are often unapproachable by any other technique. Using this method, we have discovered novel candidate genes with putative NF‐kB and apoptotic regulatory functions in Drosophila, with homologies to previously uncharacterized human genes.
Oxygen Levels and HIV‐1 Transcription
Authors: Charles Sharroya M , Ammosova Tatyana , Cardenas Jessica , Jerebtsova Marina , Niu Xiaomei , Ray Patricio , Kashanchi Fatah , Gordeuk Victor
Published: June 2021

Cellular metabolism of macrophages and T cells cultured at atmospheric 21% O2 is significantly different from cells cultured at 3–6% O2. HIV‐1 Tat induces transcription of HIV‐1 genes by recruiting host cell CDK9/cyclin T1 to TAR RNA. Our recent studies indicate that HIV‐1 transcription is also regulated by protein phosphatase‐1 (PP1). PP1 interacts with the Tat protein and disruption of this association prevents induction of HIV‐1 transcription. Here we analyzed the effect of 3% versus 21% O2 concentration on HIV‐1 transcription and replication and cellular activities of CDK9 and PP1. Using reporter assays, we found that 3% O2 inhibited HIV‐1 Tat‐induced transcription and viral replication. CDK9 activity but not CDK2 activity was inhibited in the cells cultured under 3% O2. Although PP1 expression level was not decreased, its activity was greatly reduced under 3% O2 which was due to the changes in its conformation and sensitivity to trypsin treatment. We previously showed that PP1 dephosphorylates CDK9, and decreased PP1 activity during hypoxia adversely affected CDK9 activity resulting in inhibition of HIV‐1 transcription. Our results indicate that HIV‐1 transcription is suppressed at physiological oxygen levels likely due to deregulation of host cell factors CDK9 and PP1 thus providing a new insight into the HIV‐1 latency.
Lipoteichoic acid induces HO‐1 expression via the TLR‐2/MyD88/c‐Src/NADPH oxidase pathway and Nrf2 in human tracheal smooth muscle cells
Authors: Lee I‐Ta , Wang Shyi‐Wu , Yang Chuen‐Mao
Published: June 2021

Heme oxygenase‐1 (HO‐1) is a stress‐inducible rate‐limiting enzyme in heme degradation, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. Increasing reports have indicated that lipoteichoic acid (LTA) exerts as LPS as an immune system‐stimulating agent and plays a role in the pathogenesis of severe inflammatory responses induced by Gram‐positive bacteria infection. Here, we report that LTA is an inducer of HO‐1 and examine the signaling pathways by which LTA regulates HO‐1 expression in human tracheal smooth muscle cells (HTSMCs). LTA induced HO‐1 protein, mRNA expression, and HO‐1 promoter activity. LTA‐stimulated HO‐1 expression was attenuated by transfection with dominant negative mutants of Toll‐like receptor‐2 (TLR‐2) and MyD88, the NADPH oxidase inhibitor (DPI), the ROS scavenger (NAC), and transfection with siRNAs of Src and NF‐E2‐related factor 2 (Nrf2). LTA‐stimulated c‐Src phosphorylation, translocation of p47phox and Nrf2, and ROS production were attenuated by transfection with dominant negative mutants of TLR‐2, MyD88, and c‐Src, and by pretreatment with DPI or NAC. Further studies revealed that LTA induced TLR‐2, MyD88, c‐Src, and p47phox complex formation. These results demonstrated that in HTSMCs, LTA induced ROS generation through the TLR‐2/MyD88/c‐Src/NADPH oxidase pathway, in turn initiates the activation of Nrf2, and ultimately induces HO‐1 expression.
Th17 vs. Th1 cytokine production against candida and responses to zymosan in patients with chronic airway and gut mucosal inflammation
Authors: Jyonouchi Harumi , Geng Lee , Aguila Helen A , Cushing‐Ruby Agnes , Monteiro Iona
Published: June 2021

Th17 cells appear important for defense against fungal pathogens but they can also aggravate mucosal inflammation as observed in inflammatory bowel diseases (IBD). We evaluated Th17 vs. Th1 cytokine production against candida albicans in children with airway and gut mucosal inflammation along with their responses to zymosan, a fungal derived TLR2/6 agonist. The study included children with recurrent otitis media/chronic rhinosinusitis (ROM/CRS; N=26, 17/28 diagnosed with asthma, median 6.2 y), IBD (N=18, median 13.8 y), food allergy (FA, N=41, median 3.6 y), and normal controls (N=21, median 7 y). Peripheral blood mononuclear cells (PBMCs) were tested for cytokine production with candida Ag or with zymosa. ROM/CRS and IBD but not FA PBMCs revealed higher IL‐17 production while FA and ROM/CRS but not IBD PBMCs revealed higher IFN‐γ production than controls (p<0.05). IL‐17 and IFN‐γ levels were positively correlated in FA/control cells but not in ROM/CRS/IBD cells due to higher IL‐17 production by ROM/CRS/IBD cells. With zymosan, FA PBMCs produced less IL‐10 than controls (p<0.05) and also revealed less IL‐1β production than ROM/CRS/IBD cells (p<0.01). Our results indicate aberrant Th17 vs. Th1 responses in ROM/CRS and IBD patients while FA patients may have aberrant TLR2/6 responses. Funded by Jonty Foundation/ARI.
Copyright issues: An author's perspective
Authors: Carlson Bruce M
Published: June 2021

In contemporary society, an author is not only someone who writes books, but it could also refer to someone who publishes entirely through digital media. By the same token, borrowers of illustrations or substantial portions of text could have them reproduced as hard copy or by digital means. In many respects, the copyright issues remain the same. At one level, a question involves when or should permission be obtained for an exact duplication of copyrighted material. This is straightforward when the material is to be used in a commercially‐produced medium, but the lines blur when the material is used for classroom purposes or media with small noncommercial distribution. PowerPoint presentations constitute an interesting example. Even more difficult is the issue of adapting and modifying illustrative material. At what point is copyright permission not necessary? Another tangential issue is when to contact the author as a courtesy, as well as the actual holder of the copyright. This talk will present an author's perspective on many of these issues.
Mechanical Stimulation Differentially Controls Proliferation, Morphology, and Vascular Gene and Protein Expression in Mesenchymal Progenitor Cells
Authors: Chew Douglas W. , Maul Timothy M. , Neiponice Alejandro , Vorp David A.
Published: June 2021

Introduction: Cyclic strain can regulate smooth muscle cell (SMC) gene and protein expression in mesenchymal progenitor cells (MPCs). We tested the hypothesis that the differentiation of BMPCs to endothelial cells (ECs) and SMCs may be controlled by the appropriate type, magnitude, and frequency of mechanical stimulation.

Methods: Bone marrow MPCs were seeded onto collagen I substrates and exposed to three stimuli – cyclic strain (CS), cyclic pressure (CP), steady shear stress (SS) – at 3 magnitudes and 2 frequencies for 5 days. Morphology and proliferation were analyzed by Scion Image. Protein expression was examined via immunohistochemistry. RT‐PCR was used to quantify genes relevant to various cell phenotypes and matrix proteins.

Results: MPCs responded differentially via changes in morphology, proliferation, gene, and protein expression. Cell size correlated negatively with SS and positively with CP. Proliferation was enhanced by CP, but decreased in for SS. CS caused a sequential increase in the number of SMC proteins. Although gene expression for EC and SMC genes was elevated in CP and SS, respectively, no protein expression was found. Most ECM genes were up‐regulated under CP, while only collagen I was increased in SS. Increases in TGFβ gene expression correlated with SMC and collagen I gene expression.

Conclusions: We conclude that the differentiation of MPCs towards SMCs and ECs may be regulated, in part, by the specific mechanical stimulus applied.

This work was funded by the NIH BRP grant # R01 HL069368, NIH training grant # T32 EB001026, Ruth L. Kirschstein predoctoral fellowship #F31 EB004791, and American Heart Association predoctoral fellowship #0415437U.
Embryonic myocyte‐derived factors regulate early coronary vessel development
Authors: Rodgers Laurel S , Broka Derrick M , Parker Patti , Camenisch Todd D
Published: June 2021

Many heart problems are associated with defects and disease of the coronary vessels; however coronary vessel development is not well understood. Coronary vessels originate from epicardial cells which come from the proepicardium (PE). At E9.0 in mice, PE cells migrate to and spread over the heart to form the epicardium. A subset of epicardial cells undergoes an epithelial to mesenchymal transformation (EMT) and migrates into the myocardium. These epicardial derived cells (EDC) differentiate into epithelial and smooth muscle cells of the coronary vessels. Signals required for this process are speculated to originate from the myocardium and endocardium, but there is no direct evidence for this activity. We have developed a novel in vitro system to directly address the question of “where do the signals required for early coronary vessel development originate?” In this system we co‐culture primary epicardial monolayers with embryonic myocytes. We detect an increase in epicardial EMT during co‐culture conditions compared to naïve epicardial cultures. We have identified several growth factors, including VEGF and TGF‐β2, which are produced by embryonic myocytes in vitro. Thus, our system allows for the identification of mediators responsible for epicardial EMT and, ultimately, EDC differentiation into the coronary vasculature. Supported by HL077493 and T32 HL07249.
Neural cells enhance angiogenesis in vitro through local neurotrophins secretion
Authors: Blais Mathieu , Gingras Marie , Berthod François
Published: June 2021

Nerves and blood vessels are closely associated. Moreover, sensory nerves were shown to determine the branching pattern of the vascular network in skin and to promote the arterial differentiation of blood vessels. We developed a unique in vitro model featuring a pre‐formed three‐dimensional neurites network on which a capillary‐like network was allowed to organize and mature. Sensory neurons and glial cells were co‐cultured with fibroblasts in a collagen‐chitosan sponge to reconstruct the neural network for 14 days and human endothelial cells were then seeded on the tissue to build a capillary‐like network for 17 additional days. Neural cells induced a 27% increase in the number of capillary‐like tubes (CLT) formed in the tissue. This effect was abolished when K252a, an inhibitor of the TrkA, B and C receptors for the NGF, BDNF and NT‐3 neurotrophins respectively was added to the culture medium. Moreover, we demonstrated that when 10 ng/ml of NGF, 0.1 ng/ml of BDNF, 15 ng/ml of NT3 and 50 ng/ml of GDNF were added to our endothelialized reconstructed connective tissue model, a major increase from 40 to 80% in the number of CLT was observed. This is the first in vitro demonstration of a direct angiogenic effect of peripheral neural cells on human endothelial cells through the release of neurotrophic fators and of the angiogenic potential of NT‐3 and GDNF, the latter belonging to an other family of neurotrophic factors.
Copper deficiency induces a connective tissue disorder state in a porcine native abdominal aortic aneurysm model
Authors: Sadek Mikel , Hynecek Robert L. , Poblete Honesto , Levin Elizabeth , Shin Hyun‐Joo , Lei Xingen , Faries Peter L.
Published: June 2021

Objective: Copper deficiency (CD) has been linked to impaired collagen synthesis and the development of abdominal aortic aneurysms (AAA) in swine. This study sought to evaluate the effect of a CD diet on a porcine AAA model.

Methods: A CD diet was fed to Yorkshire swine for 3mths. In CD and regular‐fed (RF) swine the infrarenal aorta was balloon dilated and infused with collagenase/elastase (16000U/1000U) (Fig 1). At 2wks, control (suprarenal aorta) and AAA tissues were frozen in liquid nitrogen and pulverized for RNA extraction. RNA from 3 CD and 3 RF samples was biotin‐labeled and hybridized to porcine arrays (20,201 genes). Relative gene intensities equaled log2(AAA/control). A 2‐class unpaired Significance Analysis of Microarray (Fig 2) test was conducted, false discovery rate (FDR) < 2.2%.

Results: 55 genes were downregulated and 20 genes were upregulated in CD compared to RF swine (Fig 3). Diminished expression of heme oxygenase and thymidilate synthetase was consistent with vascular smooth muscle and endothelial dysregulation. Differential expression of MHC classes I/II was consistent with an autoimmune etiology for AAA. Inflammatory pathology was supported by altered expressions of cytochrome P450, oligoadenylate synthetase and sphingosine kinase.

Conclusion: The addition of a CD diet to the porcine AAA model introduced novel links to known AAA connective tissue biology.

Support: NIH‐K08HL073313
Exercise Training Combined With Losartan Ameliorates Post‐infarct Ventricular Remodeling and Preserves Cardiac Function in Rats
Authors: XU XIAOHUA , WAN WENHAN , JI LISA , LAO SHUNHUA , POWERS ANTHONY S , ZHAO WEIYAN , ZHANG JOHN Q
Published: June 2021

To test the hypothesis that exercise training combined with losartan treatment after myocardial infarction (MI) could alleviate post‐MI ventricular remodeling and preserve cardiac function. Rats underwent coronary ligation or sham operation. Treatments with losartan and/or treadmill exercise were initiated 1 week post‐MI. The collagen volume in the SedMI group was significantly higher than other MI groups. Compared to the SedMI group, the hearts receiving exercise and/or losartan treatment showed significant decrease at both gene and protein levels in tissue inhibitor of matrix metalloproteinase 1 (TIMP‐1). No significant differences were found in matrix metalloproteinase (MMP) 2 or MMP‐9 among all the groups. Additionally, angiotensin II receptor type 1 (AT1) significantly decreased in response to exercise training. Furthermore, angiotensin converting enzyme (ACE) binding was markedly lower in the hearts receiving exercise training than in the SedMI hearts. Cardiac function was significantly preserved in the rats receiving exercise. Our results suggest that exercise training after MI reduces TIMP‐1 expression, and mitigates the expressions of ACE and AT1 receptor. These improvements, in turn, attenuate myocardial fibrosis and cardiac stiffness, and preserve post‐MI cardiac function. These effects are potentiated when combining exercise with losartan. Supported by NIH grant (HL074273).
SR46349B elicits head bobs in the rabbit via dopamine D1 receptors
Authors: Scarlota Laura , Harvey John , Aloyo Vincent
Published: June 2021

Rabbit head bobs are analogous to rodent head twitches and are behavioral assays of serotonin (5HT)2A receptor activation. Both are elicited by the 5HT2A/2C receptor agonist DOI and this effect is attenuated by the 5HT2A antagonist ketanserin. We have demonstrated that the 5HT2A antagonist, SR46349B (SR; Sanofi‐Synthelabo Recherche) induces rabbit head bobs which are not blocked by ketanserin thus SR does not elicit head bobs as a partial agonist at 5HT2A receptors. The current study was designed to determine if SR elicits head bobs via interaction with dopamine D1 receptors. The D1 antagonist SCH 23390 (0.05μmol/kg) was administered to New Zealand rabbits 30 min prior to DOI (0.3μmol/kg) or SR (0.26μmol/kg). In confirmation to the results obtained in rats, SCH23390 significantly attenuated DOI elicited head bobs. Similarly, SCH23390 also significantly attenuated SR elicited head bobs. SCH23390 exhibits high affinity for the D1 receptor and 25 fold lower affinity for the 5HT2A receptor. Thus, it may act as a competitive antagonist to inhibit DOI elicited head bobs. However, since ketanserin does not attenuate SR elicited head bobs, the ability of SCH23390 to interact with 5HT2A receptors cannot account for the inhibition. These results suggest DOI mediates head bobs via the expected 5HT2A receptor mechanism, but SR induces the same behavior by increasing extracellular dopamine. (MH‐16841‐39; J. A. Harvey)
The anti‐cataleptic effects of 8‐OH‐DPAT: role of pre‐synaptic 5‐HT1A receptors, and possible implications for combined treatment with antidepressants and antipsychotics
Authors: Koek Wouter , Hidalgo Jose A. , Daws Lynette C.
Published: June 2021

5‐HT1A agonists attenuate antipsychotic‐induced catalepsy, a model of extrapyramidal symptoms (EPS). To explore the role of pre‐synaptic 5‐HT1A receptors, we examined these effects in mice expressing 50% fewer serotonin transporters (5‐HTT) in which pre‐synaptic 5‐HT1A receptors are down‐regulated, but post‐synaptic 5‐HT1A receptors are not.

Mice were pretreated with a 5‐HT1A agonist (ip), treated with haloperidol (cumulative dosing), and examined for catalepsy (bar test). In C57 mice, the 5‐HT1A partial agonist buspirone did not alter the ED50 of haloperidol (0.92 mg/kg). The 5‐HT1A agonist 8‐OH‐DPAT shifted the haloperidol dose‐effect curve to the right, with 10 mg/kg producing a maximum 2.7‐fold shift. Buspirone (32 mg/kg) blocked these effects of 8‐OH‐DPAT. In 5‐HTT(+/+) mice, 10 mg/kg 8‐OH‐DPAT shifted the haloperidol dose‐effect curve 3.5‐fold to the right. In contrast, 8‐OH‐DPAT did not alter haloperidol's ED50 in 5‐HTT(+/−) mice.

The finding that 8‐OH‐DPAT had anti‐cataleptic effects in C57 and in 5‐HTT(+/+) but not in 5‐HTT(+/−) mice suggests that pre‐synaptic 5‐HT1A receptors mediate these effects. In as much as 5‐HTT(+/−) mice are a model of chronic treatment with SSRI's, the lack of anti‐cataleptic effects of 8‐OH‐DPAT in these mice suggests that SSRI's may unmask and enhance EPS of antipsychotics with 5‐HT1A activity.

Supported by NARSAD (WK) and USPHS Grant MH64489 (LCD)
Melatonin Suppresses Nighttime Memory Formation in Zebrafish
Authors: Rawashdeh Oliver , de Borsetti Nancy Hernandez , Roman Gregg , Cahill Gregory
Published: June 2021

Learning and memory are modulated by the biological clock, although the mechanisms are unknown. Here we report that in the diurnal zebrafish both learning and long‐term memory formation of an active‐avoidance conditioning paradigm occur better during the subjective day than the subjective night. Melatonin treatment during the day mimics the nighttime suppression of memory formation. Training in constant light improves nighttime memory formation, while reducing endogenous melatonin levels. Treatment with melatonin receptor antagonists at night dramatically improves long‐term memory. Removing the pineal gland, the greatest source of endogenous melatonin, also significantly improves nighttime memory formation. We adduce that melatonin is both sufficient and necessary for the poor memory formation that occurs during the subjective night.
Expression level of 5‐HT1A receptor alters firing rate and concentration‐dependency in YFP‐labeled 5‐HT dorsal raphe neurons
Authors: Metzger Kayla L , Calizo Lyngine H , Stephen Alisson , Berger Miles , Tecott Laurence H , Beck Sheryl G
Published: June 2021

The serotonin (5‐HT) 1A receptor is implicated in the etiology of anxiety and depression. Depressed suicide victims show increased expression of the 5‐HT1A receptor, and transgenic 5‐HT1A receptor knockout (KO) mice have an anxiety phenotype. Both in vivo and in vitro electrophysiological recording techniques were used to assess the effects of 5‐HT1A receptor KO and overexpression (OE) on extracelullarly recorded firing rate and 5‐HT1A receptor mediated responses. There was no difference in firing rate between KO and wild‐type (WT) mice measured extracellularly in vivo or in brain slices in which YFP labeled 5‐HT neurons were activated with 10mM phenylephrine. 5‐HT1A receptor activation with 5‐CT inhibited firing in 5‐HT neurons in raphe brain slices from WT, but not KO, mice. 5‐HT neurons from OE mice showed decreased basal firing and a leftward shift of the 5‐HT1A concentration‐response (C‐R) curve compared to WT. The 5‐HT1A receptor antagonist WAY 100635 (100nM) had no effect on basal firing rate of OE or WT neurons, but blocked the inhibition by 5‐CT. Interestingly, whole cell recordings showed no difference in the C‐R curve for 5‐HT1A receptor‐mediated outward current. These results show that KO mice have no 5‐HT neuronal response to 5‐HT1A receptor activation, whereas OE mice have increased sensitivity compared to WT depending on the active state of the neuron. Supported by MH63078.
Positive association between the mesoderm specific transcript gene and autism spectrum disorder in a Korean male population
Authors: Kwack KyuBum , Lee Seung Ku , Kim Min‐ho , Nam Min , Bang Hee Jung , Yang Jae Won , Choe Kyong‐Sik , Kim Su Kang , Hong Mee Suk , Chung Joo‐Ho , Kim Hyoun Geun
Published: June 2021

Autism spectrum disorder (ASD) is a neuro‐developmental disorder characterized by abnormalities in social, communication, and behavioral functioning. Associations between several markers in this region and ASD have been previously reported. MEST and COPG2 genes on 7q32 are logical candidate genes in this respect. In the present study, we identified associations between ASD and two adjacent genes (MEST and COPG2) in 7q32. We selected 25 single nucleotide polymorphisms (SNPs) that might be involved in expressional changes in candidate genes. Genomic DNA was collected from the venous blood samples of 147 male normal controls and 180 male Korean autism spectrum disorder patients. Genotypes of the selected SNPs were determined using Illumina Sentrix Array Matrix chips, and statistical analyses were performed using Haploview software and SAS. One SNP in the MEST gene and one haplotype involving the MEST and COPG2 genes were found to be significantly associated with ASD (both p< 0.05). ASD was found to be associated with a polymorphism in MEST, which suggests that alterations in expression of this gene may be linked to ASD susceptibility.
High Throughtput Screening Using A Polyplexed Flow Cytometry Protein Interaction Assay (FCPIA) for Small Molecule Inhibitors of Regulator of G protein Signaling (RGS) Proteins
Authors: Roman David L. , Ota Shodai , Neubig Richard R. , Ota Shodai
Published: June 2021

Signaling networks are comprised of many combinations of protein‐protein interactions and present a number of targets for potential drug discovery. Here, we focus on the interaction of Regulators or G protein signaling (RGS) proteins with the G protein Gαo, using a flow cytometery protein interaction assay (FCPIA). FCPIA is capable of accurately measuring this protein‐protein interaction and has been used in high throughput screening, resulting in the discovery of a small molecule RGS4 inhibitor (Roman, Talbot et al. 2007). We have expanded on this method by performing a high throughput screen in a polyplexed format. In this method, we attach 5 different RGS proteins (4,6,7,8 and 16) to 5 uniquely dyed bead sets and co‐incubate them with fluorescently labeled Gαo to measure the protein‐protein interaction. We established Z’ factor values for individual RGS proteins in multiplex of 0.73 to 0.92, indicating a suitable HTS method. For the screen, 5 RGS proteins on beads were combined and aliquoted into 96 well plates that contained 4 distinct compounds per well, incubated with fluorescently labeled Gαo, and then measured using FCPIA. This approach was used to screen 8,000 compounds. After individual well deconvolution, we have preliminarily identified a number of RGS and RGS‐subfamily specific novel RGS inhibitors, on which further mechanistic evaluation is underway. Supported by the NIH: DA023252 (RRN), NS057014 (RRN) and GM076821 (DLR).
RGS protein regulation and signaling diversity of protease‐activated receptors (PAR1 and PAR2)
Authors: McCoy Kelly L. , Waters Jon P. , Hepler John R.
Published: June 2021

PAR signaling must be tightly controlled since these G protein coupled receptors are proteolytically cleaved and thereby irreversibly activated. Regulators of G protein signaling (RGS) proteins provide a potential mechanism for such regulation since their conserved RGS domains give them the capacity to act as GTPase‐activating proteins and thereby inhibit G protein signaling. Little is known about RGS regulation of PAR signaling but our data using purified proteins and intact cells suggest that RGS2 and RGS4 but not RGS1 or RGS16, directly bind to and selectively regulate PAR1 and PAR2 signaling. Adding to the complexity of these studies, PAR1 and PAR2 have both been shown to couple to Gi, Gq, and G12. Our data shows that PAR1 and PAR2 activation of Gq (and potentially other G proteins) can be blocked by RGS2 and RGS4 but not by RGS1 or RGS16. Using selective G protein inhibitors and receptor mutants, ongoing studies will determine whether RGS regulation of PARs is receptor‐dependent, G protein‐ dependent or both. A detailed understanding of PAR signaling will provide insight into how these promiscuous receptors activate multiple G proteins and how their divergent signaling pathways are regulated. These studies will define PAR signaling mechanisms that underlie their roles in cellular processes such as inflammation, gliosis, and hyperalgesia. Support: R01NS037112 and R01NS049195 (JRH), A.H.A. 0715465B (KLM).
Activator of G‐protein Signaling 3 null mice: unexpected alterations in metabolic and cardiovascular function
Authors: Blumer Joe B. , Lord Kevin , Saunders Thomas L. , Black Cory , Lazartigues Eric , Varner Kurt J. , Gettys Thomas W. , Lanier Stephen M.
Published: June 2021

Activator of G‐protein Signaling (AGS) 3 and AGS5/LGN, via regulation of G‐protein signaling, play central functional roles in cell division, synaptic plasticity, addictive behavior and/or neuronal development. As part of a broad effort to define the extent of functional diversity of AGS regulated‐events in vivo, we present this report on AGS3 null mice. Surprisingly, AGS3 null adult mice exhibited unexpected alterations in cardiovascular and metabolic functions, without any obvious changes in basic behavioral traits or gross brain morphology. Despite higher food consumption AGS3−/− mice deposited significantly less fat than wild type mice, primarily due to higher rates of nocturnal energy expenditure. Conscious, unrestrained AGS3−/− mice exhibited significantly lower arterial pressures and reduced diurnal variations in arterial pressure that are associated with increased baroreceptor reflex sensitivity and a failure to adequately compensate to the decrease in arterial pressure elicited by the vasodilator sodium nitroprusside. These studies expand the functional repertoire for AGS3 and other GPR proteins providing unexpected venues for the development of therapeutic agents to influence obesity and cardiovascular regulation.
Redox Regulation of G protein βγ Subunits
Authors: Dessal Axel , Smrcka Alan V
Published: June 2021

The βγ‐subunit of G protein (Gβγ) dissociates from the α‐subunit upon activation of G protein‐coupled receptors. Once free, Gβγ is able to regulate many target proteins, including phospholipase C β2 (PLCβ2), phosphoinositide 3‐kinase γ (PI3Kγ) and G protein‐coupled receptor kinase 2 (GRK2). Our group has used a combination of virtual and manual screening to find small molecules that inhibit the interaction between Gβγ and the aforementioned proteins with affinities in the high nM range. Recently, we discovered another set of compounds (some with heavy metals, Hg2+, Cu2+ and Ag+ in their structure) that binding of a Gβγ‐binding peptide (SIGK) in an enzyme‐linked immunosorbent assay (ELISA) at concentrations in the low nM range. The effect of these small molecules persists after washing them away, and it is both prevented and reversed by dithiothreitol (DTT). This suggests that these compounds act directly at Gβγ by a reversible redox mechanism. Investigation of the mechanism of action of these compounds may lead to uncovering mechanisms for Gβγ regulation by both natural and synthetic redox active molecules.

This work was funded by National Institutes of Health GM060286
The PDZ and Band 4.1 containing protein Frmpd1 influences the subcellular location of Activator of G‐ protein signaling 3 and its interaction with G‐proteins
Authors: An Ningfei , Blumer Joe B. , Lanier Stephen M.
Published: June 2021

Activator of G protein signaling 3 (AGS3) contains seven tetratricopeptide (TPR) motifs upstream of four G‐protein regulatory (GPR) motifs, each of which can serve as a docking site for GiαGDP free of Gβγ. Such proteins have revealed unexpected functional diversity for the “G‐switch” in the control of events within the cell independent of the role of G‐proteins as transducers for G‐protein coupled receptors at the cell surface. A key question is what controls their subcellular positioning and interaction with G‐proteins. Frmpd1, which contains a PDZ and a FERM domain was identified as an AGS3‐TPR domain binding partner in yeast 2‐hybrid screens. Following co‐transfection of COS7 cells, Frmpd1 increased the amount of AGS3 associated with a membrane fraction following cell lysis. The increase in membrane‐associated AGS3 likely reflects both retention of AGS3 that is lost during membrane washing and the preferred positioning of AGS3 in a membrane fraction when complexed with Frmpd1. The interaction of AGS3 with Frmpd1 and Giα3 is mutually exclusive. Frmpd1 and AGS3 are both expressed in the neuronal catecholaminergic cell line CAD. siRNA‐mediated knock down of Frmpd1 resulted in a ~50% decrease in the amount of AGS3 associated with the membrane fraction following cell lysis. Mechanistically, Frmpd1 may stabilize AGS3 at the plasma membrane and influence the interaction of AGS3 with G protein.
Compartmentation of Cellular cGMP‐signalling: A Mechanism for Spatial and Temporal Regulation of cGMP Mediated Effects
Authors: Wilson Lindsay Shea , Elbatarny Hisham , Maurice Donald H.
Published: June 2021

Cyclic GMP (cGMP) is a ubiquitous intracellular messenger that acts to integrate and translate the information encoded by extracellular messenger molecules including nitric oxide (NO) and natriuretic peptides. Recent evidence suggests that selective activation of the NO‐sensitive guanylyl cyclase results in a localized increase of cGMP which is selectively hydrolyzed by the cGMP specific phosphodiesterase (PDE5). To investigate the role of PDE5 in maintaining this pool of cGMP, we performed selective immunoprecipitations in human platelets and discovered that PDE5 exists in two functional compartments. One compartment is soluble and enzymatically active in resting platelets, while the other fraction exists in a cGMP signaling complex with Protein Kinase G (PKG) and is not active. Interestingly, while the PKG integrated fraction of PDE5 was not activated upon addition of cGMP alone, it was markedly activated upon addition of cGMP and ATP, resulting in PKG dependent phosphorylation of Serine 92. In addition, biochemical fractionation of platelets showed that this PKG complex containing PDE5 was present on the sarcoplasmic reticulum and allows local cGMP‐mediated control of IP3‐stimualted Ca2+ release. These findings may give insight into the specific effects of nitric oxide and present new targets for sildenafil, a potent PDE5 inhibitor and active ingredient in VIAGRATM.
Angiotensin and thrombin‐induced endothelial dyfunction involves RhoA activation and elevated arginase activity
Authors: Romero Maritza Josephina , Iddings Jennifer A. , Caldwell Ruth B. , Caldwell Robert W.
Published: June 2021

Elevated arginase (ARG) activity can decrease L‐arginine availability to NO synthase (NOS) and NO production, and thus contributes to endothelial dysfunction (ED). We previously observed that increased ARG activity is involved in diabetic coronary ED in rats and mice and in impaired NO production by endothelial cells (EC) exposed to high glucose or peroxynitrite. Elevated angiotensin II (AII) and thrombin (TH) levels in diabetes are major factors contributing to its vascular complications. We tested whether activation of arginase is involved in AII or TH‐induced ED. We determined levels of ARG activity and expression and NO production (response to ionomycin) in bovine coronary EC exposed to AII or TH for 24 hrs. Both AII (10−6M) and TH (0.5 U/ml) decreased NO production by 55 and 45%, respectively, and increased ARG activity by 48% and 43%, respectively. ARG I protein expression increased by 52–58% after exposure to AII or TH. AII and TH also elevated active RhoA levels by 60 and 45%, respectively. Pretreatment with the RhoA kinase inhibitor Y‐27632 (10−5M), simvastatin (10−7M, a blocker of RhoA activation), prevented the AII‐ or TH‐ induced increases in ARG and RhoA activity and maintained normal NO production. Our results suggest AII and TH decrease NO production and cause vascular ED by mechanism(s) involving reduced L‐arginine availability for NOS via RhoA‐mediated elevations in ARG activity.
Phospholipase D2 (PLD2) and the Epidermal Growth Factor Receptor (EGFR): Identification of a Novel Relationship.
Authors: Chahal Manpreet S. , Brauner Daniel , Meier Kathryn E.
Published: June 2021

Phospholipase D2 (PLD2) catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), a lipid second messenger. PLD2 is postulated to play a role in enhancing tumorigenesis. EL4 cells, derived from a murine lymphoma, present a unique model to elucidate the role of PLD2 in signal transduction since these cells lack endogenous PLD2 mRNA and protein. In order to elucidate PLD2‐mediated signal transduction, PLD2 was stably transfected into EL4‐V7 cells. In the current study, we asked whether the EGFR, which is known to couple with PLD2, might play a role in PLD2‐mediated effects in EL4 cells. Western blotting and RT‐PCR were used to establish that both parental cells and PLD2 transfectants express endogenous EGFR. Levels of EGFR protein are increased in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. EGF stimulates proliferation of EL4 cells transfected with active PLD2, but not of parental cells or cells transfected with inactive PLD2. Inhibitor studies revealed that EGF‐induced proliferation is mediated by the PI3K/Akt pathway in cells expressing active PLD2. Active PLD2 enhances EGF‐induced invasion of cells through Matrigel, as compared to parental cells or cells expressing inactive PLD2. Taken together, the data suggest that PLD2 can act in concert with EGFR to enhance mitogenesis and invasion in lymphoma cells. (Supported by NIH CA094144‐01)
Mechanism of angiotensin II‐induced c‐Src activation in Vascular Smooth Muscle Cells
Authors: Yaghini Fariborz A , Mugabe Benon E , Buharalioglu Cuneyt K , Estes Anne M , Malik Kafait U
Published: June 2021

Angiotensin II (Ang II) activates c‐Src tyrosine kinase leading to vascular smooth muscle cells (VSMC) growth. However, the mechanism of c‐Src activation by Ang II has not yet been established. This study was performed to investigate the role of calcium (Ca2+) in Ang II‐induced c‐Src activation. Activation of c‐Src was measured by its phosphorylation using Western blot analysis and cytosolic Ca2+([Ca2+]i) by spectrofluorometry using Fura‐2. Ang II increased c‐Src phosphorylation and produced a biphasic increase in [Ca2+]i, a rapid phase followed by sustained phase. Depletion of extracellular (EC) Ca2+ or Ca2+ chelator EGTA, inhibited Ang II‐induced c‐Src phosphorylation and the sustained but not the rapid phase of [Ca2+]i. The intracellular (IC) Ca2+ chelator, BAPTA, partially reduced c‐Src phosphorylation in the presence of EC Ca2+. Infection of VSMCs with dominant negative, but not partially active c‐Src adenovirus inhibited c‐Src phosphorylation and the rapid but not the sustained phase of [Ca2+]i response to Ang II. These data suggest that Ang II promotes c‐Src phosphorylation by increasing influx of EC Ca2+. Moreover, c‐Src mediates the rapid phase of Ang II‐induced IC Ca2+ release probably via generation of inositol trisphosphate consequent to PLC activation. However, the sustained phase of cytosolic Ca2+increaseby Ang II is independent of c‐Src phosphorylation (Supported by NIH grant 079109 from HLBI).
Platelet growth factor induces smooth muscle migration through an integrin linked kinase‐dependent pathway
Authors: Esfandiarei Mitra , Yazdi Sahar Abdoli , van Breemen Cornelis
Published: June 2021

Integrin linked kinase (ILK) is a cytoplasmic interactor of integrin receptors regulating cell survival, spreading, and contraction. In this study we aimed to evaluate the role of ILK in regulating smooth muscle cell (SMCs) migration, an important event during the development of atherosclerotic plaque and coronary artery disease. Primary mouse SMCs were cultured in the presence or absence of platelet growth factor (PDGF) and various specific inhibitors. Cell lysates were used for kinase and Western blot assays. Cell migration was measured using colorimetric migration or wound healing assays utilizing fluorescent imaging. Our study showed that PDGF could activate ILK, Akt, and Erk in SMCs without any effect on focal adhesion kinase and α‐paxillin. ILK inhibition had no effect on PDGF‐induced Akt and Erk activation, indicating that their activation is through an ILK‐independent pathway. Inhibition of ILK, PI3K/Akt, and Erk1/2 decreased SMCs migration, signifying the cooperation between these signaling pathways in regulating SMC migration. To our knowledge, this is the first report of a regulatory role for ILK in SMCs migration, which is independent from Akt and Erk1/2 pathways, and conveys new insights in our efforts to establish a novel therapeutic target for treatment of coronary artery occlusion.

This work was supported by the Canadian Diabetes Association & the Michael Smith Foundation for Health Research.
Increased vascular contractile responses to phenylephrine in Doca‐salt mice is normalized by Pyk2 blockade
Authors: Giachini Fernanda R. C. , Carneiro Fernando Silva , Lima Victor Vitorino , Webb R. Clinton , Tostes Rita Cassia
Published: June 2021

Background: The calcium‐dependent proline‐rich tyrosine kinase (Pyk2), a nonreceptor protein activated by tyrosine phosphorylation, links G‐protein‐coupled receptors to vascular responses. DOCA‐salt hypertension is characterized by enhanced vascular reactivity. We tested the hypothesis that changes in vascular responses in DOCA‐salt mice are due to increased activation of Pyk2.

Methods and results: Aorta and small mesenteric arteries from DOCA‐salt and uninephrectomized (UNI) male C57bl/6 mice were incubated with vehicle or Tyrphostin A‐9 (0.1μM, Pyk2 inhibitor). Systolic blood pressure (mmHg) was higher in DOCA (126±3) vs UNI (100±4). Vascular responses to PE (1nM to 10mM) were greater in vessels from DOCA‐salt than UNI, but treatment with Tyrphostin A‐9 abolished the difference among the groups (Figure 1). Pyk2 levels, as well as Pyk2Tyr402 phosphorylation, paxillin and paxillinTyr118 phosphorylation were increased in DOCA‐salt aorta (Figure2). Incubation with Tyrphostin A‐9 restored phosphorylation of Pyk2 and paxillin.

Conclusion: Increased Pyk2 expression and activity are involved in increased vascular contractile‐responses in Doca‐salt mice.

Financial Support: NIH (HL‐74167, RCW); FAPESP and CAPES, Brazil.
Identification of the alpha‐2 adrenoceptor subtype causing contraction of rat lateral tail vein
Authors: Yao Mingyi , Abel Peter W
Published: June 2021

Constriction of veins plays an important role in thermoregulation. Norepinephrine from sympathetic nerves can activate alpha‐2 adrenoceptors in veins causing constriction and reduced heat loss in response to cooling. We determined the alpha‐2 adrenoceptor subtype causing contraction of the rat lateral tail vein studied in vitro. Tail veins were isolated, endothelium removed, and incubated in Krebs solution at 37 degrees C. Concentration‐response curves for contraction caused by the alpha‐2 adrenoceptor agonist UK 14304 were generated in the absence and presence of the alpha‐2 adrenoceptor antagonists RX 821002 (non‐subtype selective), BRL 44408 (alpha‐2A selective), ARC 239 (alpha‐2B/C selective) and MK 912 (alpha‐2C selective). UK 14304 caused concentration‐dependent contraction of the tail vein with an EC50 of 8.3 nM. The affinities (KB) of antagonists in blocking UK14304 contraction were RX 821002 (2.3 nM), BRL 44408 (64 nM), ARC 239 (199 nM) and MK 912 (0.06 nM). The affinity of MK 912 was nearly the same as its affinity for alpha‐2C adrenoceptors reported by others, and affinities for other antagonists were consistent with alpha‐2C adrenoceptors. Thus, alpha‐2C adrenoceptors mediate UK 14304 induced contraction of the rat lateral tail vein. The development of alpha‐2C selective agents may be useful to selectively regulate venous function in diseases associated with altered thermoregulation.
Increased serum adiponectin by heme oxygenase‐1 in obese diabetic zucker rats increases insulin sensitivity and glucose tolerance
Authors: Kim Dong Hyun , Burgess Angela P. , Peterson Stephen J. , Li Ming , Asprinio David , Abraham Nader G.
Published: June 2021

We hypothesized that heme oxygenase‐1 (HO‐1) attenuates diabetic complications via stimulation of a potent vascular protector including adiponectin in rodent with type 2 diabetes. We examined the effect of cobalt protoporphyrin (CoPP) on serum levels of adiponectin in obese and obsess‐diabetic Zucker rats. The onset of diabetes or obesity coincided with a decrease in HO activity, which was restored by administration of CoPP. Upregulation of HO‐1 expression in diabetic rats produced an increase in adiponectin (p<0.003), decreased superoxide (p<0.05) and reduced blood pressure (p<0.01). Increased HO activity was associated with an increase in insulin sensitivity, glucose tolerance and significant reduction in body weight and fat content. The increases in HO‐1‐adiponectin resulted in a decrease within TNF, IL‐1 and IL‐6. The effect of HO‐1 on human MSC show similar results, increases in HO‐1 and decreased adipogenesis. The administration of an inhibitor of HO activity decreased adiponectin. This study suggests that the anti‐diabetic/anti‐obesity effect of HO‐1 is due to regulation of adipogenesis, increases adiponectin secretion, decreases TNF, IL‐1 and IL‐6. HO‐1 treatment was associated with the restoration of insulin sensitivity, preserve pancreatic insulin response to glucose, reduction in weight gains and the amelioration vascular diseases. This work is supported by NIH grants HL55601 and HL34300.
Effects of Angiotensin II on CaV1.2 Expression in Mesenteric Artery
Authors: Wang Wenze , Palade Philip
Published: June 2021

Effects of angiotensin II (Ang II) on CaV1.2 L‐type calcium channel expression in 24 h cultured mesenteric arteries (MA) of Sprague‐Dawley rats were studied using Western blot, real‐time RT‐PCR and blood vessel tension recording. Ang II dose‐dependently upregulated expression of CaV1.2 protein but not its mRNA. Ang II still increased CaV1.2 expression with protein synthesis inhibited by puromycin. The upregulation of CaV1.2 expression by Ang II was blocked by protein kinase C (PKC) inhibitor chelerythrine, by NAD(P)H oxidase inhibitors diphenyleneiodonium (DPI) or apocynin, or by endothelial damage by brief perfusion with 0.3% CHAPS. The Ang II upregulation of CaV1.2 expression was blocked by catalase, and H2O2 dose‐dependently upregulated CaV1.2. After endothelial damage, H2O2 still upregulated CaV1.2 expression and this effect was inhibited by the PI3 kinase inhibitor wortmannin. MA cultured in Ang II displayed increased tension in response to CaV1.2 channel agonist FPL 64176. These results suggest that: (1) decreased degradation of CaV1.2 channel protein may contribute to Ang II‐induced upregulation of CaV1.2 expression, (2) Ang II activation of PKC, NAD(P)H oxidase and H2O2 production in the endothelium and H2O2 activation of PI3 kinase in the vascular smooth muscle mediate the upregulation, and (3) the upregulation of CaV1.2 protein contributes to increased vascular tone. Supported by HL 63903.
ULTRASTRUCTURAL BASIS OF ASYNCHRONOUS URIDINE 5′‐TRIPHOSPHATE (UTP)‐INDUCED CA(2+) WAVES IN RAT BASILAR ARTERY
Authors: Syyong Harley , Yang Clarice , Trinh Gina , Kuo Kuo‐Hsing , van Breemen Cornelis
Published: June 2021

A common form of Ca2+ signaling in smooth muscle cells is Ca2+ oscillations. We observed that uridine 5′‐triphosphate (UTP) elicits cyclic, dose‐dependent increases in intracellular Ca2+ which underlie tonic force generation in rat basilar artery, similar to those observed in conduit arteries. We also observed subplasmalemmal junctions between the plasma membrane (PM) and sarcoplasmic reticulum (SR), known as PM‐SR junctions. Pharmacological dissociation of these junctions abolishes maintenance of Ca2+ waves, but does not affect Ca2+ release. The maintenance of Ca2+ waves is dependent on extracellular Ca2+ entry, which is mediated by L‐type voltage‐gated calcium channels (L‐VGCCs), non‐selective cation channels (NSCCs), and by the plasma membrane Na+/Ca2+ exchanger (NCX) working in reverse‐mode. Blockade of inositol 1,4,5‐triphosphate receptors (IP3Rs), but not ryanodine receptors, abolishes ongoing Ca2+ waves. These findings suggest that Ca2+ waves in rat basilar artery underlie tonic force generation and are due to repetitive cycles of Ca2+ release from the IP3Rs, and that extracellular Ca2+ influx through L‐VGCCs, NSCCs, and reverse‐mode NCX is important for their maintenance by supplying Ca2+ for SR refilling. Finally, despite its similarity to resistance vessels, the basilar artery behaves like a conduit artery with respect to the mechanism of Ca2+ waves. This work was funded by CIHR of Canada.
Apocynin reduces infarct volume following cerebral ischemia in mice
Authors: Jackman Katherine A , Miller Alyson A , De Silva T Michael , Drummond Grant R , Sobey Christopher G
Published: June 2021

We tested the effect of apocynin, an NADPH oxidase inhibitor, on cerebral artery production of superoxide (O2‐) and hydrogen peroxide (H2O2), and on stroke outcome in C57Bl6/J and Nox2 KO mice. Apocynin (300 μM) inhibited NADPH‐induced O2‐ production by ∼40% (n=9, P<0.05), and reduced H2O2 levels by ∼80% (n=6, P<0.05), in cerebral arteries. Interestingly, apocynin also reduced detectable H2O2 by >80% in the absence of arterial tissue. Ischemic stroke was induced by 30 min middle cerebral artery occlusion (n=79). Mice received vehicle (0.1% DMSO), 2.5 or 5 mg/kg apocynin ip 30 min before ischemia. Mortality at 24 h was 12% in vehicle controls, and 0% (P=0.24) and 41% (P<0.05) in mice treated with 2.5 and 5 mg/kg apocynin, respectively. Infarct volume was reduced by 2.5 mg/kg apocynin by ∼50% (n=8; P<0.05), whereas 5 mg/kg apocynin was ineffective (n=13). Apocynin (2.5 mg/kg) had no effect on infarct volume in Nox2 KO mice, suggesting its protective effect is Nox2‐dependent. Thus, apocynin inhibits cerebral NADPH oxidase, directly scavenges H2O2, and has a narrow therapeutic window for stroke. As H2O2 is an endogenous cerebral vasodilator, high dose apocynin could compromise blood flow.