Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation

We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA–mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cell, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53

An investigation into the mechanisms of tissue factor-mediated apoptosis in endothelial cells

Cells are known to express and release tissue factor (TF) following activation. In addition, endothelial cells are capable of acquiring TF carried by circulating microvesicles. Accumulation of TF in the endothelium contributes to chronic pathological conditions including cardiovascular disease. The aim of this study was to investigate the mechanisms that regulate the release of TF within microvesicles. In addition, the effects of accumulation of TF on the mediators of cell proliferation and apoptosis were examined and the underlying mechanisms explored. Throughout this study, endothelial cells were transfected to express TF or, alternatively, incubated with TF-containing microvesicles to permit accumulation of TF within cells. Activation of PAR2 receptor in TF-bearing cells resulted in prolonged activation of p38 which was enhanced by preventing the phosphorylation of Ser253 within the cytoplasmic domain of TF through Ala-substitution. Moreover, the inhibition of p38α resulted in decreased Ser258 phosphorylation and increased TF release as microvesicles. Expression of wild-type TF or Asp253-substituted TF induced cell proliferation via a mechanism that involves the up-regulation of Cyclin D1. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, but only following cell activation. The level of p53 protein, p53-phosphorylation at Ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing the wild-type and Ala253-substituted TF. These observations were reversed by inhibition of p38α using either SB202190 or siRNA-mediated suppression. The expression of bax and p21 mRNA and Bax protein was also increased in cells expressing Ala253-substituted TF, but not in cells expressing wild-type TF. Inhibition of transcriptional activity of p53 with pifthrin-α or inhibition of p38α suppressed the expression of bax. These data suggest that the activation of endothelial cells expressing TF prolongs p38α activation which in turn phosphorylates TF at Ser258 and terminates TF release within microvesicles. This leads to the accumulation of TF within cells and can induce cell apoptosis. The mechanism of apoptosis is mediated via the activation of p38α which in turn phosphorylates p53 at Ser33 and stabilises p53 within the nucleus. This study has shown a mechanism associating increases in circulating TF-containing microvesicles with endothelial depletion and the progression of vascular disease

A Randomized, Controlled, Double-Blind Study of Light Emitting Diode Photomodulation for the Prevention of Radiation Dermatitis in Patients with Breast Cancer

Background and objectivesRadiation dermatitis occurs in a majority of patients with breast cancer who receive radiation therapy (RT), causes significant pain, and may necessitate treatment delay. Light emitting diode (LED) photomodulation has been reported to minimize radiation dermatitis. This study sought to further evaluate the efficacy of LED photomodulation in lessening radiation dermatitis.Materials & methodsAfter surgery, patients with breast cancer received LED photomodulation or sham treatments in conjunction with three-dimensional conformal RT. Reactions were evaluated using standardized photographs graded according to National Cancer Institute criteria.ResultsIn the LED treatment group (n=18), no patients had grade 0 reactions, six (33.3%) had grade 1 reactions, 12 (66.7%) had grade 2 reactions, and none had a grade 3 reaction. In the sham treatment group (n=15), one (6.6%) patient had a grade 0 reaction, four (26.7%) had grade 1 reactions, 9 (60.0%) had grade 2 reactions, and one (6.7%) had a grade 3 reaction. Two (11.1%) patients in the LED treatment group and one (6.7%) in the control group had to interrupt treatment. Differences between groups were not statistically significant.ConclusionLED photomodulation did not reduce the incidence of radiation-induced skin reactions or interruptions in therapy.

Photobinding of Triflusal to Human Serum Albumin Investigated by Fluorescence, Proteomic Analysis, and Computational Studies

[EN] Triflusal is a platelet antiaggregant employed for the treatment and prevention of thromboembolic diseases. After administration, it is biotransformed into its active metabolite, the 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). We present here an investigation on HTB photobinding to human serum albumin (HSA), the most abundant protein in plasma, using an approach that combines fluorescence, MS/MS, and peptide fingerprint analysis as well as theoretical calculations (docking and molecular dynamics simulation studies). The proteomic analysis of HTB/HSA photolysates shows that HTB addition takes place at the epsilon-amino groups of the Lys137, Lys199, Lys205, Lys351, Lys432, Lys525, Lys541 and Lys545 residues and involves replacement of the trifluoromethyl moiety of HTB with a new amide function. Only Lys199 is located in an internal pocket of the protein, and the remaining modified residues are placed in the external part. Docking and molecular dynamic simulation studies reveal that HTB supramolecular binding to HSA occurs in the "V-cleft" region and that the process is assisted by the presence of Glu/Asp residues in the neighborhood of the external Lys, in agreement with the experimentally observed modifications. In principle, photobinding can occur with other trifluoroaromatic compounds and may be responsible for the appearance of undesired photoallergic side effects.We gratefully acknowledge financial support from the Spanish Government (CTQ2016-78875-P, SAF2016-75638-R, BES-2014-069404, and RETICS network ARADyAL RD16/0006/0030), the Generalitat Valenciana (PROMETEO/2017/075 and CIDEGENT/2018/044), the Xunta de Galicia [Centro Singular de Investigacion de Galicia accreditation 2016-2019 (ED431G/09), ED431B 2018/04 and post-doctoral fellowship to EL], and the European Union (European Regional Development Fund-ERDF). The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF. We are grateful to the Centro de Supercomputacion de Galicia (CESGA) for use of the Finis Terrae computer.Molins-Molina, O.; Pérez Ruiz, R.; Lence, E.; González-Bello, C.; Miranda Alonso, MÁ.; Jiménez Molero, MC. (2019). Photobinding of Triflusal to Human Serum Albumin Investigated by Fluorescence, Proteomic Analysis, and Computational Studies. Frontiers in Pharmacology. 10:1-9. https://doi.org/10.3389/fphar.2019.01028S1910Cuquerella, M. C., Lhiaubet-Vallet, V., Cadet, J., & Miranda, M. A. (2012). Benzophenone Photosensitized DNA Damage. Accounts of Chemical Research, 45(9), 1558-1570. doi:10.1021/ar300054eDarden, T., York, D., & Pedersen, L. (1993). Particle mesh Ewald: AnN⋅log(N) method for Ewald sums in large systems. 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The Pharmacokinetic Changes in Cystic Fibrosis Patients Population: Narrative Review

Cystic fibrosis (CF) is a rare genetic disorder commonly affecting multiple organs such as the lungs, pancreas, liver, kidney, and intestine. Our search focuses on the pathophysiological changes that affect the drugs’ absorption, distribution, metabolism, and excretion (ADME). This review aims to identify the ADME data that compares the pharmacokinetics (PK) of different drugs in CF and healthy subjects. The published data highlight multiple factors that affect absorption, such as the bile salt precipitation and the gastrointestinal pH. Changes in CF patients’ protein binding and body composition affected the drug distribution. The paper also discusses the factors affecting metabolism and renal elimination, such as drug–protein binding and metabolizing enzyme capacity. The majority of CF patients are on multidrug therapy, which increases the risk of drug–drug interactions (DDI). This is particularly true for those receiving the newly developed transmembrane conductance regulator (CFTR), as they are at a higher risk for CYP-related DDI. Our research highlights the importance of meticulously evaluating PK variations and DDIs in drug development and the therapeutic management of CF patients