Inhibition of Ocular Tumor and Endothelial Cell Growth with a TEAD4216 Peptide Fragment

Purpose: Transcriptional enhancer factor 1-related (RTEF-1) also known as TEAD4 is expressed in ocular vascular endothelial cells and plays a role in the control of VEGF expression. Alternative processing of TEAD4 hnRNA results in different proteins able to stimulate or inhibit VEGF gene transcription. The purpose of this study is to test whether short peptide fragments (STY-RMR), representing functional domains of the inhibitory TEAD4216 isoform, can inhibit tumor as well as endothelial cell proliferation.Experimental Design: Cell proliferation was assessed using a colorimetric assay in cell lines incubated with STY-RMR, the amount of secreted VEGF within media was determined both in treated and control cell lines.Results: Significant dose dependent inhibition of cell proliferation was observed. Maximal inhibition of ocular melanoma (Mel 202 and Mel 207) cell proliferation was observed at a dose of 30 mg/100ml of STY-RMR (87% and 60% inhibition, respectively). At the same dose, more than 50% inhibition was observed in retinoblastoma and breast cancer cells (P <0.001). Significant inhibition of primate ocular endothelial cell proliferation (42% at 30 mg/100 ml (p < 0.001), and retinal pigment epithelial cells showed also a 75% inhibition (p = 0.007). Secreted VEGF was decreased in the media of all tested cell lines that had been exposed to STY-RMR.Conclusion: Functional short peptide domains derived from the TEAD4216 isoform may prove to be useful for treatment of ocular tumors and other VEGF dependent neovascular disease.Inhibition of proliferation and VEGF production within ocular endothelial cells indicate the potential of this agent to treat age-related macular degeneration (ARMD) and diabetic retinopathy (DR)

Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells

© 2018 Royal Australian and New Zealand College of Ophthalmologists. This author accepted manuscript is made available following 12 month embargo from date of publication (January 2018) in accordance with the publisher's archiving policy.Background Over‐production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS‐generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. Methods Differential Nox enzyme expression was studied by real‐time‐quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2′,7′‐dichlorofluorescein. DMOG‐stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. Results Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG‐induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. Conclusions Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases

ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells

© The Author(s) 2018 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Abstract Objective Regulation of intercellular adhesion molecule (ICAM)-1 in retinal endothelial cells is a promising druggable target for retinal vascular diseases. The ICAM-1-related (ICR) long non-coding RNA stabilizes ICAM-1 transcript, increasing protein expression. However, studies of ICR involvement in disease have been limited as the promoter is uncharacterized. To address this issue, we undertook a comprehensive in silico analysis of the human ICR gene promoter region. Results We used genomic evolutionary rate profiling to identify a 115 base pair (bp) sequence within 500 bp upstream of the transcription start site of the annotated human ICR gene that was conserved across 25 eutherian genomes. A second constrained sequence upstream of the orthologous mouse gene (68 bp; conserved across 27 Eutherian genomes including human) was also discovered. Searching these elements identified 33 matrices predictive of binding sites for transcription factors known to be responsive to a broad range of pathological stimuli, including hypoxia, and metabolic and inflammatory proteins. Five phenotype-associated single nucleotide polymorphisms (SNPs) in the immediate vicinity of these elements included four SNPs (i.e. rs2569693, rs281439, rs281440 and rs11575074) predicted to impact binding motifs of transcription factors, and thus the expression of ICR and ICAM-1 genes, with potential to influence disease susceptibility. We verified that human retinal endothelial cells expressed ICR, and observed induction of expression by tumor necrosis factor-α

The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases

E-Letter responding to 'Increased serum levels of the chemokine CXCL13 and up-regulation of its gene expression are distinctive features of HCV-related cryoglobulinemia and correlate with active cutaneous vasculitis' by Sansonno et al

E-Letter available from site of original article only. http://www.bloodjournal.org/content/112/5/1620.e-lettersCXCL13 – also known as BLC in the mouse and BCA-1 in the human – is a chemokine with high selectivity for B lymphocytes. In seeking primers for detection of human CXCL13, we read the report by Sansonno et al. This work describes expression of CXCL13 in hepatitis C virus (HCV)-associated mixed cryoglobulinemia.This work was supported in part by research funding from the National Eye Institute/National Institutes of Health (R21EY022009 to JRS); and the Australian Research Council (FT130101648 to JRS)

MOESM2 of ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells

Additional file 2: Table S2. Predicted transcription factor binding sites within genome evolutionary rate profiling (GERP)-constrained elements

MOESM4 of ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells

Additional file 4: Table S4. Primer pairs and product sizes for gene transcripts studied in human retinal endothelial cells. References are provided for primers sequences sourced from the literature

MOESM3 of ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells

Additional file 3: Table S3. Description of phenotype-nucleotide polymorphisms (SNPs) predicted to influence transcription of the human ICR gene

MOESM1 of ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells

Additional file 1: Table S1. Sources for eutherian genomes used in genome evolutionary rate profiling (GERP) and chromosomal locations of GERP-constrained elements

A randomised controlled trial of the effect of a connected inhaler system on medication adherence in uncontrolled asthmatic patients

Suboptimal adherence to maintenance therapy contributes to poor asthma control and exacerbations. This study evaluated the effect of different elements of a connected inhaler system (CIS), comprising clip-on inhaler sensors, a patient-facing app and a healthcare professional (HCP) dashboard, on adherence to asthma maintenance therapy. This was an open-label, parallel-group, 6-month, randomised controlled trial in adults with uncontrolled asthma (asthma control test (ACT) score less than 20) on fixed-dose inhaled corticosteroids/long-acting β-agonist maintenance therapy (n=437). All subjects received fluticasone furoate/vilanterol ELLIPTA dry-powder inhalers for maintenance and salbutamol/albuterol metered-dose inhalers for rescue, with a sensor attached to each inhaler. Participants were randomised to one of five CIS study arms (allocation ratio 1:1:1:1:1) reflecting the recipient of the data feedback from the sensors, as follows: 1) maintenance use to participants and HCPs (n=87); 2) maintenance use to participants (n=88); 3) maintenance and rescue use to participants and HCPs (n=88); 4) maintenance and rescue use to participants (n=88); and 5) no feedback (control) (n=86). For the primary endpoint, observed mean±sd adherence to maintenance therapy over months 4–6 was 82.2±16.58% (n=83) in the “maintenance to participants and HCPs” arm and 70.8±27.30% (n=85) in the control arm. The adjusted least squares mean±se was 80.9±3.19% and 69.0±3.19%, respectively (study arm difference: 12.0%, 95% CI 5.2–18.8%; p<0.001). Adherence was also significantly greater in the other CIS arms versus the control arm. The mean percentage of rescue medication free days (months 4–6) was significantly greater in participants receiving data on their rescue use compared with controls. ACT scores improved in all study arms with no significant differences between groups. A CIS can improve adherence to maintenance medication and reduce rescue medication use in patients with uncontrolled asthma