bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantifying binding of VEGF‐A isoforms at VEGFR2 in membranes
Authors: Peach Chloe J , Kilpatrick Laura E , Woolard Jeanette , Hill Steve J 
Published: June 2021

Vascular endothelial growth factor A (VEGF‐A) is a key mediator of angiogenesis, primarily signalling via VEGF Receptor 2 (VEGFR2) [1]. There are numerous endogenous isoforms of VEGF‐A resulting from alternative mRNA splicing. We previously used bioluminescence resonance energy transfer (BRET) to quantify binding of VEGF‐A isoforms single‐site labelled with tetramethylrhodamine (TMR) at NanoLuciferase (NanoLuc)‐tagged VEGFR2 in living cells [2,3]. However, VEGFR2 undergoes ligand‐dependent and ‐independent endocytosis, therefore we aimed to compare the pharmacology of VEGF‐A isoforms at VEGFR2 in membrane preparations. Membranes were homogenised from HEK293T cells stably expressing NanoLuc‐VEGFR2 as in [4], stored in Phosphate Buffered Saline and plated on the day of experimentation as 5μg/well in 40μl vehicle (Hanks Buffered Saline Solution/0.1% Bovine Serum Albumin; pH 7.4). For kinetic experiments, membranes were incubated with the NanoLuc substrate furimazine (10μM) for 5min, then 1–20nM fluorescent VEGF‐A was added. BRET ratios were measured every 30sec for 90min at 37°C using a BMG Pherastar. To quantify binding affinities, membranes were incubated with increasing concentrations of VEGF165a‐TMR, VEGF165b‐TMR or VEGF121a‐TMR (0.1–20nM) in the presence and absence of 100nM unlabelled VEGF. Alternatively, membranes were co‐stimulated with increasing concentrations of unlabelled VEGF‐Ax and a fixed concentration of fluorescent VEGF‐A (0.5–5nM). Following 60min stimulation at 37°C, furimazine was added and BRET ratios were measured. The kinetic binding profiles of each fluorescent VEGF‐A isoform in membranes reached a plateau at NanoLuc‐VEGFR2 and binding was maintained for 90min. This contrasted with the characteristic decrease in BRET ratio observed following 20min in intact cells [2]. Each fluorescent VEGF‐A isoform had saturable binding at NanoLuc‐VEGFR2, with a similarly low level of non‐specific binding compared to cells [3]. Derived equilibrium dissociation constants in membranes showed VEGF165a‐TMR, VEGF165b‐TMR and VEGF121a‐TMR bound NanoLuc‐VEGFR2 with nanomolar affinity. Each fluorescent VEGF‐A isoform was displaced by unlabelled VEGF‐Ax, from which nanomolar affinities were derived for VEGF‐Ax. Using NanoBRET to monitor protein‐protein interactions therefore showed that while the kinetic binding profile was strikingly different in membrane preparations compared to intact cells, derived affinity constants were identical. This quantitative approach retains the ability for receptor oligomerisation in an intact membrane bilayer, while isolating molecular ligand/receptor interactions at 37°C without the complex spatio‐temporal dynamics governing full‐length VEGFR2 in living cells.Support or Funding InformationThis project is supported by the British Pharmacological Society's AJ Clark Scholarship.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 Circulating EGF‐like Ligands on NADPH Oxidase Expression in Vascular Cells
Authors: Aitchison Alexandra H , Lucitti Jennifer L , Miller Francis J 
Published: June 2021

The mechanisms responsible for the increased risk of cardiovascular disease in patients with chronic inflammatory conditions are not clear. We and others have shown that the vascular NADPH oxidases have an important role in mediating vascular disease. Expression of the pro‐atherogenic Nox1 NADPH oxidase is regulated in cultured smooth muscle cells (SMC) by activation of the epidermal growth factor receptor (EGFR). We previously reported that epidermal growth factor‐like ligands (EGF‐LL) are increased in serum and vessels of a non‐human primate model of atherosclerosis. We hypothesized that increased circulating EGF‐LL in chronic inflammatory conditions enhance vascular disease via increased NADPH oxidase expression and activation. Cultured primary human aortic SMC were serum deprived and treated with the EGF‐LL amphiregulin (AREG), epiregulin (EREG), epidermal growth factor (EGF) and heparin‐binding EGF‐like growth factor (HB‐EGF) and NADPH oxidase expression measured by qPCR. Among the EGF‐LL, AREG was most effect at increasing Nox1 expression (4.5‐fold increase compared to control) in SMC, followed by HB‐EGF (1.7‐fold increase compared to control). We observed marked variability or no change of expression of the other NADPH oxidase subunits, including Nox4, Nox5, p22phox, NoxA1, NoxO1, p47phox, p67phox. EGF‐LL had no significant effect on the expression of NADPH oxidase in human EC, although there was variability in experiments. Because we previously showed that Nox1 activation contributes to cytokine activation of NFκB in SMC, we used immunohistochemistry to evaluate TNFα stimulated nuclear translocation of p65 in mouse and human SMCs exposed to EGF‐LLs. TNFα stimulated p65 activity to a much greater degree in human SMCs compared to mouse, but activity was unchanged by pretreatment with EGF‐LL. To better emulate in vivo conditions, we harvested aortic segments from C57BL/6 (WT) and Nox1−/− mice. Segments were exposed to either AREG, a pro‐inflammatory cytokine mix of tumor necrosis factor (TNFα) and interleukin 1 beta (IL‐1β), or both. Exposure to AREG for 24 hours increased Nox1 expression in WT (2.3‐fold). Ongoing studies are examining the response in Nox1−/− mice. In summary, these data suggest that circulating AREG may contribute to development of vascular disease by increasing SMC Nox1 expression and identify circulating EGF‐LL as potential therapeutic target in patients with chronic inflammation.Support or Funding InformationFJM is supported by the Office of Research and Development, Department of Veterans Affairs [2I01BX001729] and the National Institutes of Health [HL130039].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Inflammatory Signaling via Interleukin‐13 Decreases Potassium Channel Expression and Increases Vascular Smooth Muscle Excitability
Authors: Sandhu Gurpreet , Brueggemann Lyubov , Wolfgang Riley , Cribbs Leanne L. , Byron Kenneth L. 
Published: June 2021

The transcriptional repressor element‐1 (RE1) silencing transcription factor (REST), also known as neuron‐restrictive silencer factor (NRSF) is a gene silencing transcription factor, which has been characterized as a master regulator of the neuronal phenotype. REST plays a key role in the development of the nervous system and is a critical player in the etiology of neurodegenerative diseases such as Huntington's Disease, though little is known about the role of REST in non‐neuronal cells. We are exploring a potential role of REST as a mediator between inflammation and changes in smooth muscle phenotype. Kv7.5 voltage‐dependent potassium channels, encoded by the KCNQ5 gene, are expressed in smooth muscle cells where they play an important role in determining resting membrane potential. Interleukin‐13 (IL‐13) is an inflammatory cytokine, which is implicated as a primary mediator of the phenotypic changes induced by allergic inflammation in many tissues. In the present study, we hypothesized that IL‐13 can increase nuclear REST and decrease KCNQ5 mRNA expression and Kv7.5 protein in vascular smooth muscle cells, providing a potential link between inflammation, REST and potassium channel‐mediated vascular hyper‐responsiveness. In support of this hypothesis, using A7r5 rat aortic smooth muscle cells, we found that 1) IL‐13 treatment significantly increased nuclear REST, and 2) significantly suppressed the expression of KCNQ5 and Kv7.5 currents, resulting in increased sensitivity of A7r5 cells to arginine vasopressin (AVP). 3) REST overexpression also significantly decreased Kv7.5 currents, and 4) significantly increased the sensitivity of A7r5 cells to AVP. We conclude that inflammation that induces IL‐13 may increase nuclear REST levels and thereby decrease KCNQ5 expression and Kv7.5 currents, ultimately increasing vascular smooth muscle cell excitability and contributing to vascular hyper‐responsiveness.Support or Funding InformationThe project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circulating NRG‐1b does not contribute to plasma ErbB3‐activating activity
Authors: Boateng Emmanuel , deKay Joanne T , Peterson Sarah M , Robich Michael P , Sawyer Douglas B , Ryzhov Sergey 
Published: June 2021

Neuregulin‐1b (NRG‐1b) is a membrane‐bound or secreted growth and differentiation factor that mediates its action by binding to ErbB receptors. Circulating levels of NRG‐1b are characterized by large inter‐individual variability with the range of absolute values covering two orders of magnitude, from hundreds to tens of thousands of picograms per milliliter of blood. NRG‐1b signaling via ErbB receptors contributes to the survival of endothelial and other cells and downregulation of the inflammatory response. A higher level of circulating NRG‐1b may indicate increased expression and secretion or shedding of membrane‐bound NRG‐1b, which in turn can contribute to better protection against cardiovascular stress or injury. However, it is unknown whether circulating NRG‐1b can induce activation of ErbB receptors.Here, we performed an analysis of circulating NRG‐1b functional activity using a cell‐based ELISA to measure ErbB3‐activating activity in blood plasma obtained from healthy donors and subjects undergoing coronary artery bypass grafting (CABG) surgery. Activation of ErbB3 receptors, expressed at high level in the MCF‐7 breast cancer cell line, was determined by measuring ErbB3 phosphorylation (Tyr1289).High levels of plasma ErbB3‐activating activity were detected in healthy donors and CABG subjects. However, no correlations were found between the levels of plasma NRG‐1b and ErbB3‐activating activity. To determine the direct effect of circulating NRG‐1b we incubated blood plasma with an NRG‐1 neutralizing antibody, which prevented the stimulatory effect of recombinant NRG‐1b on activation of ErbB3 receptors. No effect of neutralizing antibody was found on the ErbB3‐activating activity of plasma obtained from healthy donors or CABG subjects. Analysis of plasma before and after CABG revealed a significant decrease in the levels of ErbB3‐activating activity but not circulating NRG‐1b at 24 hours after surgery.Our results demonstrate that circulating NRG‐1b cannot induce ErbB3 activation and emphasize the importance of functional testing of NRG‐1b proteins in biological samples. Further investigation of post‐translational modifications of circulating NRG‐1b is warranted.Support or Funding InformationThis work was supported by the Maine Medical Center Cardiovascular Research Institute 2015 Pilot Project Program, the National Heart, Lung, and Blood Institute of the National Institutes of Health under grant R01 HL136560 and the American Heart Association under grant 17POST33410474.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Calcium Sensitization in 5‐Hudroxytryptamine‐Induced Contraction of Rat Aorta with or without Perivascular Adipose Tissue
Authors: Oriowo Mabayoje , Al‐Jarallah Aishah , Oommen Elsie 
Published: June 2021

We have previously shown that the perivascular adipose tissue (PVAT) exerted an anti‐contractile effect against 5‐hydroxytryptamine (5‐HT)‐induced contraction of aorta segments from pregnant and non‐pregnant rats. This anti‐contractile effect was significantly reduced in aorta segments from pregnant rats. In the present investigation, we have examined the possible role of calcium sensitization involving ROCK and PKC in this loss of anti‐contractile effect of PVAT. Both Y‐27632 (10−6M) and GF 109203X (10−6M), inhibitors of calcium sensitization, significantly reduced 5‐HT‐induced contractions of PVAT‐free aorta segments from non‐pregnant and pregnant rats. However, they were less effective in aorta segments from pregnant rats. PVAT reduced the effectiveness of Y‐27632 and GF 109203X in aorta segments from pregnant and non‐pregnant rats. ROCK‐I and ROCK‐II protein were expressed in aorta segments and PVAT from pregnant and non‐pregnant rats. There was a reduction in the expression of both isoforms in aorta segments but not PVAT from pregnant rats. In addition, there was no significant difference in the expression of ROCK‐I and ROCK‐II in PVAT from pregnant and non‐pregnant rats. We concluded that the loss of anti‐contractile effect of PVAT in aorta segments from pregnant rats could not be explained by altered calcium‐sensitization mechanisms.Support or Funding InformationResearch Sector, Kuwait University, KuwaitThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Ferrochelatase For Treating Of Retinal Neovascularization
Authors: PASHA SHEIK PRAN BABU SARDAR , CORSON TIMOTHY W 
Published: June 2021

BackgroundPathological retinal neovascularization (RNV) is a common microvascular complication in several retinal diseases including retinopathy of prematurity, proliferative diabetic retinopathy, and central retinal vein occlusion. RNV is currently treated by inhibition of vascular endothelial growth factor (VEGF), with some therapeutic success, but some patients do not respond to anti‐VEGF treatment, calling for additional means to block RNV. We previously identified the heme biosynthesis enzyme ferrochelatase (FECH) as an angiogenesis mediator. FECH inhibition is, therefore, a promising therapeutic approach for blocking RNV in the eye. Here, we investigated the inhibition of FECH using chemical and genetic approaches in the mouse oxygen‐induced retinopathy (OIR) model of RNV.MethodsIn the OIR model, newborn wild‐type or Fechm1Pas mice (with a partial loss‐of‐function mutation) are exposed to 75% oxygen at postnatal day 7 (P7) for 5 days, causing retinal vascular regression. At P12, upon return to normoxia, the retina becomes ischemic, forming RNV. Vascular pathology was assessed at P17 using Isolectin B4 staining. In a chemical approach, FECH inhibitor N‐methyl protoporphyrin (NMPP), was intravitreally injected in OIR mice at P12 and RNV assessed at P17. Pimonidazole hydrochloride and 5‐ethynyl‐2′‐deoxyuridine (EdU) were administered intraperitoneally at different time points to monitor hypoxia and cell proliferation in retinal tissue. Temporal expression of FECH was analyzed at various ages in mouse OIR retinas using immunoblot and immunostaining.ResultsRetinal flat mounts of the OIR model, which features central avascular retina, exhibit co‐localization of hypoxic, pimonidazole‐adduct immunostaining and neovascular tufts across the retina using Isolectin B4 immunostaining. Temporal FECH upregulation was observed in mouse OIR retinas by immunoblot, and immunostaining revealed that FECH is colocalized with neovascular tufts. A partial loss of function allele in the Fech m1Pas mouse significantly reduced pathological RNV (****P<0.0001, N=8–15 eyes) and cell proliferation (****P<0.0001, N=4–6 eyes) in the OIR model compared to wild‐type littermates. In another approach, chemical inhibition of FECH by NMPP treatment significantly suppressed the RNV and cell proliferation (****P<0.0001, N=8 eyes) and also significantly promoted physiological angiogenesis compared to vehicle treatment in the OIR mouse model.ConclusionsMutation of Fech and chemical inhibition using NMPP reduces pathological RNV and intriguingly, also promotes physiological angiogenesis, suggesting a dual effect on vascular repair upon FECH inhibition. Together, these findings suggest that FECH inhibitors could be used to treat neovascularization in ocular diseases.Support or Funding InformationNIH/NEI R01EY025641This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ERICH3 Characterization: Function in Vesicular Trafficking and Antidepressant Treatment Response
Authors: Liu Duan , Zhuang Yongxian , Neavin Drew , Zhang Lingxin , Kaddurah‐Daouk Rima , Wang Liewei , Weinshilboum Richard 
Published: June 2021

Major depressive disorder (MDD), the number one psychiatric disease and a major cause of medical disability worldwide, is a heterogeneous disease. Efforts to identify biomarkers for antidepressant therapy response by the use of genome‐wide association studies (GWAS) have generally yielded disappointing results, e.g. only a few markers have been replicated across studies. By applying a “pharmacometabolomics‐informed pharmacogenomic” research strategy, we previously identified a genome‐wide significant single‐nucleotide polymorphism (SNP) signal in the glutamate‐rich 3 (ERICH3) gene that was associated with plasma serotonin concentrations which were themselves associated with selective serotonin reuptake inhibitor (SSRI) response in MDD patients enrolled in the Mayo Clinic PGRN‐AMPS SSRI trial. The same ERICH3 SNPs were associated with SSRI response in other GWAS, including the STAR*D and ISPC studies. However, the function of ERICH3 and the molecular mechanism(s) by which it might be associated with plasma serotonin concentrations and with SSRI response remain unknown. Functional characterization of ERICH3 and its encoded protein might make it possible to identify novel mechanisms of antidepressant treatment response. In the present study, ERICH3 mRNA and protein were studied in cell lines from human peripheral blood and brain. ERICH3 cDNA constructs and specific antibodies were generated to make it possible to study its function. Two ERICH3 mRNA splice variants and protein isoforms, one “full‐length” and the other encoding a C‐terminal protein, were identified in a variety of human cell lines and in human brain tissue. ERICH3 mRNA expression and immunofluorescent staining suggested both cytosolic and vesicular localizations for ERICH3 as well as co‐localization with serotonin and dopamine in both mouse and human neurons. Co‐immunoprecipitation studies identified several ERICH3 interacting proteins, including PIK3C2A, CLTC and AP2A2, proteins known to play a role in vesicular trafficking, as well as ALK and CUX1, both of which display genetic polymorphisms that have been associated with SSRI response in GWAS studies of antidepressant treatment response in MDD patients. This series of experiments suggests that ERICH3 may play a role in vesicular trafficking and, as a result, may influence neurotransmitter actions in the brain that are associated with antidepressant treatment response.Support or Funding InformationThis work was supported by the NIH grants U19 GM61388, R01 GM28157 and T32 GM008685.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beta‐cell‐specific loss of the inhibitory G protein, Gαz, alters development and pathophysiology of Type 1 Diabetes
Authors: Fenske Rachel , Peter Darby , Wienkes Haley , Reuter Austin , Carbajal Kathryn , Kimple Michelle 
Published: June 2021

The mechanisms that underlie the beta‐cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our lab has discovered a role for the unique inhibitory G‐protein, Gαz, in beta‐cell function, survival, and replication in both normal conditions and those of pathological stress. Gαz, when acting as a canonical inhibitory G‐protein, reduces production of cAMP through inhibition of adenylate cyclase. Cyclic AMP acts as essential player in the amplification of glucose‐stimulated insulin secretion, while also mediating pathways that regulate beta‐cell death and replication. Previous work in our lab has shown that non‐obese diabetic (NOD) mice with global loss of Gαz are protected from developing T1D‐like hyperglycemia through increased beta‐cell function and replication and decreased beta‐cell death. In order to determine if Gαz in the beta‐cell was solely responsible for the protected phenotype, we generated a beta‐cell‐specific Gαz knockout by breeding Gz Flox/Flox mice with Rat Insulin Promoter (RIP) driven CreHerr mice, on the NOD background for more than 10 generations. Experimental mice were sNP genotyped to ensure preservation of NOD loci. Weekly random‐fed blood glucose measurements were taken beginning at weaning. Greater than 75% of Gαz β‐cell‐KO females remained euglycemic over the first 30 weeks of life compared to 61% in Cre+ NOD controls. Despite no difference in fasting blood glucose at either 12 or 28 weeks of age, Gαzβ‐cell‐KO females exhibited improved glucose tolerance as compared to Cre+ controls; a phenotype independent of increased baseline insulin or stimulated insulin secretion. Beta‐cell area is equivalent between Gαzβ‐cell‐KO males and sex‐matched Cre+ controls. Most interestingly, insulitis is dramatically reduced with loss of beta‐cell Gαz, suggesting a role for Gαz− in mediating inflammatory processes driving T1D pathophysiology. Additionally, Gαzβ‐cell‐KO animals have increased alpha‐cell active glucagon‐like peptide 1 (GLP‐1), a known promoter of beta‐cell function and survival. Experiments are ongoing to further investigate other facets of beta‐cell health, including function, and to delineate important communication pathways between islet cell types. Thus far, our results show a critical role for beta‐cell Gαz in promoting T1D pathophysiology as a hub of intra‐islet cell‐cell signaling.Support or Funding InformationJDRF J&J Industry Partnership Award 17‐2011‐608; NIH‐NIDDK R01 DK102598 and F31 DK109698‐01A1; VA BLR&D I01 BX003700‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.