bluuebunny/crossref_metadata_2025 huggingface dataset:
Venetoclax, a Novel BCL‐2 Inhibitor, Induces Cell Growth Suppression, Apoptosis, Cell Cycle Arrest, and Autophagy in Triple Negative Breast Cancer MDA‐MB‐231 Cells
Authors: Korashy Hesham M. , Alatwi Fahad O. , Alhoshani Ali , El Gamal Heba M. , Shamoon Licia S. , Ghanim Sarah S. , Agouni Abdelali
Published: June 2021

Venetoclax (VCX), formally known as ABT‐199, is a selective and orally bioavailable small‐molecule B‐cell lymphoma 2 (BCL‐2) Homology 3 (BH3) mimetic, with potential pro‐apoptotic and antineoplastic activities. In April 2016, VCX was approved in the USA to treat patients with chronic lymphocytic leukemia (CLL) with chromosome 17p deletion who have received at least one prior treatment. VCX inhibits the activity of a novel target, Bcl‐2 protein, liberating the BH3‐only proteins and thus restoring apoptotic processes in tumor cells, via activation of BAK/BAX‐mediated apoptosis. The BCL‐2 protein is commonly expressed in several of human cancer including solid tumors, such as breast cancer, ovarian, lung, colorectal, stomach, and prostate cancers, as its expression has been shown to be associated with an aggressive disease course and poor survival. Although, BCL‐2 is expressed in several sub‐types of breast cancer, neither VCX nor any other BCL‐2 inhibitor agents alone or in combination with other chemotherapeutic agents have been tested against triple negative breast cancer (TNBC). Accordingly, we hypothesized that VCX is able to induce TNBC cell growth inhibition via induction of apoptosis, autophagy, and cell cycle arrest. To test this hypothesis, human TNBC MDA‐MB‐231 cells were treated for indicated intervals with increasing concentrations of VCX, thereafter, the effects of VCX on cell proliferation and cell cycle arrest were determined using flow cytometry. Furthermore, the apoptogenic effect of VCX was explored by measuring the percentage of apoptotic cells using flow cytometry and the expression of caspases, BAX, and BCl2 genes at the mRNA, protein, and activity levels. The results of the present study showed that VCX decreased the growth and proliferation of TNBC MDA‐MB‐231 cells in a concentration manner. This growth suppression was associated with a significant increase in the percentage of apoptotic MDA‐MB‐231 cells with a significant increase in the expression of several apoptotic genes, caspase3, caspase7, and BAX in concentration‐dependent manner. Importantly, the VCX‐induced‐apoptosis was accompanied with cell cycle arrest at G0/G1 phase with a concentration‐dependent inhibition of cell cycle proliferator genes, such as Cyclin D1 and E2F1. In addition, VCX treatment induced a significant increase in the level of autophagy marker LC3‐II mRNA and protein expression levels. In conclusion, the present study demonstrates the first evidence that VCX induces human TNBC MDA‐MB‐231 cell growth inhibition through the induction of apoptosis, cell cycle arrest, and autophagy mechanisms.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Sensitivity of Two Triple Negative Breast Cancer Cell Lines to 2, 3‐ dichloro‐5, 8‐dimethoxy‐1, 4‐naphthoquinone and 4 Hydroxy‐Tamoxifen
Authors: Robinson Anastasia G.J. , Copeland Robert Leon
Published: June 2021

Breast cancer (BC) is a heterogeneous disease and it is one of the leading causes of mortality worldwide in women each year. However, in the United States, there is a disparity in survival between African American (AA) and Caucasian women with AA women having a higher rate of mortality. BC is typically classified based on the expression of the following receptors: Estrogen receptor α (ERα), Progesterone receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER2). Conventional treatment for BC relies on the expression of ERα for anti‐estrogenic therapy and HER2 for HER2 inhibition. Triple negative breast cancer (TNBC) is a form of breast cancer which lacks expression of ERα, PR and HER2 and affects minority population disproportionately. Potential mechanisms for the loss of ERα expression include hyperactivation of MAPK pathways due to EGFR or HER2 overexpression, estrogen withdrawal, hypoxia and methylation of the CpG island, cytosine‐guanine abundant area, on the ER gene promoter region. Due to lack of targetted treatment for TNBC, alternative treatment options are essential to increase patient survivability. 2, 3‐ dichloro‐5, 8‐dimethoxy‐1, 4‐naphthoquinone (Z285), a 1,4 napthoquinone analog, has demonstrated cytotoxic effects in estrogen positive and estrogen negative cell lines. Previous in silico data showed that this compound may function as an MAPK allosteric inhibitor. MCF7 cells were used as an ERα positive control and two TNBC cell lines, HCC1806, from a AA woman, and MDA MB 231, from a Caucasian woman, were used. Cell viability studies were carried out on cells treated with Z285 alone (0.5–10μM), 4 Hydroxy‐Tamoxifen (4OH‐Tam) alone (3–30μM) for 24,72 and 120hrs. In combination studies, the cells were pretreated with Z285 (2 or 5μM) for 6 hrs followed by 4OH‐Tam (1–15μM) for 24, 72 and 120hrs. These results showed a dose and time‐dependent response with individual treatment. Combinatory studies showed significant decreases in cell viability when compared to individual studies. Cells were treated with 5 μM Z285 and mRNA was collected for RNA microarray analysis to determine changes in mRNA expression profile. These results showed modulation of several genes involved in the MAPK pathway and suggest a mechanism of increased sensitivity of the TNBC cells to 4OH‐Tamoxifen. Therefore, this novel compound which targets the MAPK pathway may offer a model for treating TNBC breast cancers by reactivating ERα expression in these cancers and thus rendering them susceptible to anti‐estrogen therapy.

Support or Funding Information

Charles and Mary Latham Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeted Osmotic Lysis of H28 Mesothelioma Cells
Authors: Edenfield Samantha , Sherman Kelly Jean , Scahill Steven D. , Gould Harry J. , Paul Dennis
Published: June 2021

Mesothelioma is an aggressive cancer of the pleura cells of the chest cavity commonly linked to asbestos exposure. With a post‐diagnosis prognosis of between 6 and 12 months. Traditional chemotherapy increases survival time by only 11 weeks. Targeted Osmotic Lysis (TOL) is a novel cancer therapeutic that exploits cancer cells' upregulation of Voltage‐Gated Sodium Channels (VGSC) by pairing a pharmacological blocker of Na+, K+‐ATPase (sodium pumps), such as digoxin or ouabain, with electric or magnetic stimulation of VGSCs. The increase in intracellular Na+ causes an osmotic lysis of cells that over‐express VGSCs. TOL has been shown to selectively lyse highly invasive breast cancer cells, such as MDA‐MB 231, while leaving the normal tissue intact. To test the hypothesis that TOL would be an effective treatment for mesothelioma, we incubated H28 mesothelioma cells or MeT‐5A normal pleural effusion cells in 0–500 nM digoxin for 10 min, then activated the VGSCs with a 0–5 VDC electric current for 30 min. Using video microscopy, lysis was assessed by two independent observers with cytosolic extrusion as the measure. TOL effectively lysed H28 cells dose‐ and stimulus‐dependently, with a maximum cell death of 94.2% Less than 5% of cells in the Drug only, Stimulation only, and Non‐treated groups died (p<.0001). MeT‐5A cells did not lyse. Likewise, when we used a pulsed magnetic field (90 mT, 25 pps) to stimulate VGSCs, H28 cells lysed dose‐ and stimulation‐dependently, whereas control treatments and Met‐5a cells did not. Together these results are evidence that TOL causes significant cell death in mesothelioma cells compared to the normal cells. Thus, TOL may be an effective treatment for mesothelioma.

Support or Funding Information

This research was supported by a grant from Oleander Medical Technologies

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Impact of RGS6 on GIRK‐dependent Muscarinic and Adenosine Receptor Signaling in Mouse Sinoatrial Nodal Cells
Authors: Anderson Allison , Wickman Kevin
Published: June 2021

Introduction

Parasympathetic slowing of heart rate is largely mediated by muscarinic receptor (M2R) activation of the G protein gated inwardly‐rectifying potassium (GIRK) channel on sinoatrial nodal (SAN) cells and atrial myocytes. The strength of M2R‐GIRK signaling is negatively regulated by regulator of G protein signaling 6, RGS6. Genetic ablation of RGS6 in mice gives rise to enhanced M2R‐GIRK signaling in SAN cells, resulting in both exaggerated M2R‐induced bradycardia and increased susceptibility to pacing‐induced atrial fibrillation. Adenosine, used clinically to both diagnose and treat certain arrhythmias, exerts these actions through adenosine receptor (A1R) activation of cardiac GIRK channels. The extent to which RGS6 regulates A1R‐GIRK signaling, however, is not understood. Here, we investigated and compared the impact of RGS6 on A1R‐GIRK and M2R‐GIRK signaling in mouse SAN cells and heart rate.

Methods and Results

Using whole‐cell patch‐clamp electrophysiology, we measured the efficacy and potency of M2R‐ and A1R‐GIRK channel signaling in adult SAN cells from wildtype and Rgs6−/− mice. SAN cells from Rgs6−/− mice displayed both prolonged channel deactivation kinetics and increased channel sensitivity to CCh‐induced currents as compared to wild‐type SAN cells. Surprisingly, we found no difference in kinetics or channel sensitivity of A1R‐GIRK responses in Rgs6−/− SAN cells. We did observe a striking, significant increase in the amplitude of the A1R‐GIRK response in Rgs6−/− SAN cells compared to wild‐type controls. Recordings from mice lacking cardiac GIRK channels (Girk4−/−) confirm that M2R‐ and A1R‐induced currents are GIRK‐dependent. Furthermore, occlusion studies in wild‐type SAN cells suggest that M2R activates nearly all of the GIRK channels present in SAN cells, while A1R activates a smaller portion of GIRK channels. Intriguingly, RGS6 ablation seems to allow for the activation of a larger proportion of GIRK channels by A1R. Heart rate measurements from isolated Rgs6−/− hearts show exaggerated bradycardia in response to the same doses of CPA, an A1R agonist, as compared to wild‐type controls. Significantly lower heart rates were observed in response to lower doses of CCh in isolated Rgs6−/− hearts as compared to wild‐type hearts, suggesting a shift in sensitivity.

Conclusions

Our results suggest that A1R‐GIRK channel signaling displays distinct differences from M2R‐GIRK channel signaling, resulting in distinct negative regulation by RGS6 in mouse SAN cells and the isolated heart.

Support or Funding Information

This work was supported by NIH grants KW (HL105550) and AA (HL139090).

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

This 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 Information

FJM 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 Information

The 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 Information

This 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 Information

Research Sector, Kuwait University, Kuwait

This 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

Background

Pathological 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.

Methods

In 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.

Results

Retinal 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.

Conclusions

Mutation 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 Information

NIH/NEI R01EY025641

This 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 Information

This 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 Information

JDRF J&J Industry Partnership Award 17‐2011‐608; NIH‐NIDDK R01 DK102598 and F31 DK109698‐01A1; VA BLR&D I01 BX003700‐01A1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neutrophil GPCRs as novel therapeutic targets
Authors: Corriden Ross
Published: June 2021

Each year in the United States, 2 million people are infected with antibiotic resistant pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA); 23,000 patients die as a result of such infections. With the growth of antibiotic resistance and the slowing in the development of new antibiotics, the discovery of new, effective approaches to treat infections is an important unmet medical need. G protein‐coupled receptors (GPCRs) in polymorphonuclear neutrophils (PMNs), the first‐line effectors of the innate immune response, are a novel but unexplored potential class of “immune boosting” targets. Using a combination of qPCR based arrays and RNAseq analysis, we defined and quantified GPCR expression in human PMNs and found 174 GPCRs commonly expressed among healthy donors tested. To help determine which GPCRs might be involved in response to pathogens, we assessed the GPCR profile of PMNs incubated with MRSA for 3 h. We found numerous GPCRs whose expression increased at least 2‐fold upon pathogen exposure. Analyses of PMN function (e.g., chemotaxis, extracellular trap production, bacterial killing) using selective agonists/antagonists of several such GPCRs indicated that pharmacological agents can “boost” the function of PMNs, including the killing of bacteria. The results indicate that: 1) Human PMNs express a large profile of GPCRs, expression of a number of which is altered by exposure to pathogenic bacteria. 2) Previously unrecognized PMN‐expressed GPCRs can boost PMN function, including the ability to kill bacteria. These data thus suggest a new paradigm for enhancement of innate immunity: targeting of neutrophil‐expressed GPCRs as a means to treat bacterial infections, including ones caused by antibiotic‐resistant bacteria. Furthermore, such GPCRs may also represent new therapeutic targets for the treatment of inflammatory disorders driven by aberrant neutrophil function.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
1α,25‐Dihydroxy vitamin D3, the active form of vitamin D3, protects against allergeninduced airway constriction
Authors: Maarsingh Harm , Remick Adam J , Stimson David TJ , Lopez Alejandro , Bolson Anthony , DellaVecchia Matthew
Published: June 2021

Allergen‐induced bronchoconstriction is an important hallmark of allergic asthma and is caused by the release of mediators, such as histamine, by activated mast cells. In various disease models, the vitamin D3 pathway has been shown to reduce mast cell degranulation. Vitamin D3 is converted into 25‐hydroxy vitamin D3 and subsequently into the active form 1α,25‐dihydroxy vitamin D3 by CYP enzyme activity. Vitamin D3 deficiency contributes to the pathophysiology of allergic diseases, including asthma, whereas vitamin D3 treatment reduces allergen‐induced airway hyperresponsiveness and inflammation in experimental models of asthma.

In this study, we investigated the role of the vitamin D3 pathway in regulating allergen‐induced constriction of intrapulmonary airways in an animal model of allergic asthma.

To this aim, precision cut lung slices (PCLS) were obtained from guinea pigs that were IgE‐sensitized to ovalbumin (OVA) and sacrificed 4–8 weeks later. Lungs were filled with 1.5% agarose, cooled with ice, harvested and 500‐μm thin PCLS were prepared. Constrictions of the intrapulmonary airways to increasing concentrations of OVA or histamine were monitored using video‐assisted microscopy. PCLS were pre‐treated with or without vitamin D3 compounds 30 minutes prior to the addition of OVA or histamine.

OVA induced a dose‐dependent and full constriction of intrapulmonary airways in guinea pig PCLS. Pretreatment with 1α,25‐dihydroxy vitamin D3 (1 μM), the active form of vitamin D3, reduced the sensitivity (pD2) of the airways to OVA by more than 300‐fold (p<0.01), without affecting the maximal constriction (Emax). By contrast, the bronchoconstriction induced by exogenous histamine was not affected by pretreatment with 1α,25‐dihydroxy vitamin D3. Taken together, these findings indicate that the bronchoprotective effect of 1α,25‐dihydroxy vitamin D3 is caused by inhibiting the allergen‐induced release of mast cell mediators rather than by reducing the responsiveness to these mediators.

In conclusion, treatment with 1α,25‐dihydroxy vitamin D3 reduces the airway sensitivity towards allergens and subsequent airway constriction, presumably by reducing allergen‐induced mast cell degranulation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐allergic Effect of Dan phenolic acid A from Salvia miltiorrhiza in Ovalbumin‐induced Asthma Model
Authors: Son So‐Eun , Heo Jae‐Yeong , Im Dong‐Soon
Published: June 2021

Background

In Oriental countries, the root of Salvia miltiorrhiza is used to treat cardiovascular diseases, asthma, and other conditions. Dan phenolic acid B from S. miltiorrhiza extracts has been shown to improve airway hyperresponsiveness. We aimed to investigate the effects of Dan phenolic acid A and Dan Shen ketone from S. miltiorrhiza in allergic asthma using female Balb/c mice and rat RBL‐2H3 mast cells.

METHODS

To elucidate whether Dan phenolic acid A, a constuent of Salvia miltiorrhiza extract has beneficial effects on allergic asthma, anti‐asthma effects were studied using female Balb/c mice and rat RBL‐2H3 mast cells. Antigen‐induced degranulation was assessed by measuring b‐hexosaminidase activity in vitro. In addition, a murine ovalbumin‐induced allergic asthma model was used to test the in vivo efficacy of Dan phenolic acid A and Dan Shen ketone.

RESULTS

Antigen‐induced degranulation was inhibited by Dan phenolic acid A and Dan Shen ketone. Sensitization and challenge of ovalbumin induced allergic asthma responses. Administration of Dan phenolic acid A and Dan Shen ketone decreased numbers of inflammatory cells, especially eosinophils, and reduced the expression and secretion of Th2 cytokines, including IL‐4 and IL‐13, in lung tissues and bronchoalveolar lavage fluid. Histologic studies showed administration of Dan phenolic acid A and Dan Shen ketone reduced inflammatory signs and mucin production in lungs.

CONCLUSION

These findings provide evidence that Dan phenolic acid A and Dan Shen ketone have a potential use as anti‐allergic therapeutics.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exercise Training‐MicroRNA‐126 Upregulation is Associated with Prevention of Dexamethasone‐Mediated Microvascular Rarefaction
Authors: Herrera Naiara Araújo , de Souza Francine Duchatsch Ribeiro , Tardelli Lidieli Pazin , Dionisio Thiago José , dos Santos Carlos Ferreira , Vasquez‐Vivar Jeannette , do Amaral Sandra Lia
Published: June 2021

Dexamethasone (DEX) treatment often increases blood pressure. Exercise training is an efficient non‐pharmacological intervention to treat hypertension, which is thought to be explained by improved microvascular function. Microcirculation related‐microRNAs (miRNAs) play a role in maintaining micro vessel skeletal density. The aim of this study was to investigate if training‐induced miRNAs are associated with exercise training‐dependent prevention of DEX‐induced rarefaction. Wistar rats were trained (treadmill) for 8 weeks or not (sedentary) and then treated with DEX (50 μg/kg per day, s.c.) or saline for 14 days. Carotid artery catheterization for arterial pressure measurements was performed on last day of treatment and tibialis anterior (TA) muscle was collected for morphometric, protein and microRNAs analyses. In sedentary rats, DEX treatment induced hypertension (+27%) concomitantly with capillary density loss (CD, −20.8%) and decreased VEGF (−43.0%), p‐AKT/AKT (−39.6%) and Bcl‐2 (−23.0%) protein levels and increased caspase‐3‐cleaved protein (+34.0%) in TA muscle. In the trained group, however, microRNA‐126 expression was increased (+13.1%), which was followed by enhanced p‐AKT/AKT (+37.7%) and Bcl‐2 (+7.7%), as well as a diminished caspase‐3‐cleaved (−23.1%) protein level. The changes in miRNA‐126 expression were positively correlated with p‐AKT/AKT (r=0.7246) and VEGF (r=0.5224) protein levels. Neither DEX nor training significantly changed miRNA‐16, 21, 221, 222, 155. We concluded that miRNA‐126 upregulation, induced by training, plays an important role in controlling skeletal muscle vessel density. MiRNA126 could be a novel therapeutic agent against DEX‐induced rarefaction.

Support or Funding Information

Financial support: FAPESP (2018/06998‐7)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ADAM17 impairs F11R/JAM‐A‐mediated wall shear stress mechanosensing and induces aging‐related inward artery remodeling
Authors: Tian Yanna , Czikora Istvan , Bagi Zsolt
Published: June 2021

We sought to determine if lumen narrowing inward arterial remodeling is mediated by impaired endothelial mechanosensing of wall shear stress (WSS) in aging. Previously we have shown that aging upregulates A disintegrin and metalloproteinase ADAM17 in the vascular endothelium. ADAM17 sheds F11R/JAM‐A, an important tight junction‐located endothelial cell (EC) adhesion molecule. Here we tested if ADAM17 activation promotes aging‐related inward artery remodeling by shedding F11R/JAM‐A. Human arteries were obtained from fat biopsies in 40 consecutive patients. We found that vascular ADAM17 enzyme activity increases significantly with age, whereas F11R/JAM‐A expression was reduced in older (>64 yo) subjects, which was associated with reduced luminal diameter and increased wall thickness. Cultured human ECs exposed to increasing levels of flow (0 to 8 dyne/cm2) progressively changed their orientation to the direction of flow. In the presence of recombinant ADAM17 or after siRNA‐mediated silencing of F11R/JAM‐A, ECs failed to efficiently orient with the direction of flow. Gene transfer of ADAM17 cleavage resistant, mutated JAM‐A(V227Y) resulted in a normal EC orientation to flow. Collectively, our results uncover a novel regulatory role for vascular ADAM17 in reducing F11R/JAM‐A‐dependent WSS mechanosensing, which can lead to lumen narrowing inward arterial remodeling in aging.

Support or Funding Information

NIH R01AG054651

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 Insight Into the Determinants of Skeletal Muscle Oxygen Consumption by Dual‐Wavelength Diffuse Correlation Spectroscopy
Authors: Rosenberry Ryan , Tucker Wesley J. , Trojacek Darian , Chamseddine Houda , Arena‐Marshall Carrie , Tian Fenghua , Haykowsky Mark J. , Nelson Michael D.
Published: June 2021

Near‐infrared diffuse correlation spectroscopy (DCS) is a promising new optical imaging technique to noninvasively quantify microvascular perfusion. Combining DCS with conventional near‐infrared spectroscopy (NIRS) provides novel insight into the determinants of skeletal muscle oxygen consumption, by measuring microvascular oxygen delivery (by DCS) and oxygen utilization (by NIRS, i.e. tissue saturation). To test this hypothesis, we compared DCS‐derived measures of skeletal muscle oxygen delivery and utilization against Doppler ultrasound and direct measures of oxygen saturation from the venous effluent of the exercising muscle. Eight healthy male subjects (age 27.2 ± 4.2; BMI 26.9 ± 4.3) performed a graded handgrip exercise test. Subjects were instructed to perform rhythmic hand grip exercise on a two‐second duty cycle, at 10, 13, 16, and 19 kg. All subjects were instrumented with the DCS probe placed over the belly of the flexor digitorum profundus, a retrograde venous catheter (deep vein) inserted into the antecubital vein, and Doppler ultrasound over the brachial artery. Heart rate was monitored by ECG and blood pressure by continuous finger‐pressure of the non‐gripping hand. The major findings were two‐fold: First, we observed a strong intra‐individual relationship between DCS‐derived muscle perfusion and Doppler‐derived brachial artery blood flow (r2 = 0.95). Second, we observed an unexpected dissociation between skeletal muscle saturation, as measured by DCS, which declined progressively with each incremental workload (BL 71.9 ± 0.29%, 10kg 68.4 ± 0.91%, 13 kg 64.2 ± 1.04%, 16kg 61.8 ± 0.54%, 19kg 59.4 ± 1.26%), while directly measured venous oxygen saturation declined with the onset of exercise but remained unchanged thereafter (BL, 65.3 ± 7.20%; 10kg, 39.8 ± 8.42%; 13 kg, 39.5 ± 7.83%, 16kg, 42 ± 4.14%, 19kg, 41 ± 3.38%). Taken together, these results support DCS as a valuable tool, capable of providing novel insight into the determinants of skeletal muscle oxygen consumption at the microvascular level. The fact that tissue saturation was dissociated from venous saturation suggests that DCS may provide insight beyond conventional measurements.

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