77 research outputs found
Linking Induction and Transrepression of PPARĪ²/Ī“ with Cellular Function
The copyrights of all papers published in this journal are retained by the respective authors as per the 'Creative Commons Attribution License' (http://creativecommons.org/licenses/by/3.0/).Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors and members of the nuclear hormone receptor superfamily. PPARĪ²/Ī“ is ubiquitously expressed and has a central role in homeostasis, and has been suggested as a therapeutic target for a number of metabolic and cardiovascular disorders. This important nuclear receptor controls transcription under different modes of molecular activity which directly control the cellular function and fate of tissues. This complex activity of induction and transrepression of gene expression (with and without exogenous ligands) is poorly understood and yet understanding this molecular control through novel drug development would led to control over a key molecular switch in all cells. This review outlines the main molecular mechanisms of PPARĪ²/Ī“, and links the modes of activity to the signalling pathways in inflammation, proliferation and senescence, with the goal to understand how this will translate into novel drug design to control the PPARĪ²/Ī“ molecular switch.Peer reviewe
Effects of Apamin and Charybdtoxin on Endothelium Independent Vasodilatation : Implications in the study of EDHF
Endothelial derived hyperpolarizing factor (EDHF), together with endothelial derived NO and prostacyclin represent the major endogenous vasodilator hormone pathways (Coleman et al, 2004). Since the chemical nature of EDHF is uncertain, many studies have relied on pharmacological tools. Often EDHF described as the endothelial dependent dilation which is resistant to a combination of drugs that inhibit NO synthase (usually L-NAME) and cyclo-oxygenase (usually indomethacin) but abolished by those that block large and small Ca2+-activated K+ channels (BKCa and SKCa; with charybdotoxin plus apamin respectively). This approach relies on each of these drugs being selective for endothelial dependent responses. In the current study we have taken a different approach to most and performed experiments where inhibitors were added to endothelium denuded vessels after dilators induced stable vasodilatation. Male Wistar rats (200 Ā± 15.4g) were killed by lethal exposure to CO2. Second order mesenteric arteries were mounted in isometric wire myographs and vasomotor responses recorded as described previously (Harrington and Mitchell, 2004). In some experiments vessels were contracted with EC80 concentration of U46619, dilation was then induced by the addition of 3x10-6M acetylcholine or 3x10-6M SNP. When either L-NAME (10-4M) or charybdotoxin (10-7M) plus apamin (5x10-7M) were added after acetylcholine the vasodilatation was immediately and completely reversed (Figure A and B respectively). In other experiments, endothelium independent vasodilation induced by SNP, were also reversed by charybdotoxin plus apamin, but not L-NAME. Figure A. Example trace of an artery re-contracting following the addition of L-NAME in pre-contracted arteries dilated with acetylcholine. Bar Graphs show re-contraction of arteries in response to L-NAME or charybdotoxin plus apamin (C+A) in arteries with endothelium, dilated with acetylcholine (Figure B) or without endothelium dilated with SNP (Figure C). This data demonstrates that charybdotoxin and apamin have pharmacological effects independent of the endothelium, at the level of smooth muscle cell function. These observations suggest results obtained with these drugs in relation to EDHF studies, should be treated with caution. Harrington L and Mitchell JA (2004) Br J Pharmacol 143: 611-617 Coleman et al (2004) Clin Ex Pharm Phys 31; 641-649Peer reviewedFinal Accepted Versio
The āEDHFā Antagonist 14, 15 Epoxyeicosa-5(Z)-Enoic Acid has Vasodilator Properties in Mesenteric Vessels
There is now overwhelming evidence for Epoxyeicosatrienoic acids (EETs) as endothelial derived hyperpolarising factor (EDHF). Most recently, a number of pharmacological tools have been developed for the study of EETs in relation to EDHF responses. EETs have been shown to cause relaxation by activating smooth muscle large conductance Ca2+ sensitive K+ (BKCa) (Archer et al, 2003). This dilatory response has been shown to be specifically inhibited by its analogue 14, 15-epoxyeicosa-5 (Z) enoic acid (14, 15 EEZE) in both human internal mammary artery and bovine coronary artery (Archer et al, 2003). Here we have investigated the antagonist effects of 14, 15 EEZE in murine arteries. Male Black 6 mice (12-18 weeks) were killed by lethal exposure to CO2. First order arteries were isolated and mounted in wire myographs immersed in physiological salt solution (PSS). Arteries were equilibrated (30 mins) and tensions normalised as described previously (Mulvany and Halpern, 1977). Arteries incubated for 30 minutes with or without 3Āµg/ml 14, 15 EEZE. A concentration response curve to 11, 12 EET was performed cumulatively on arteries pre-contracted with EC80 U46619. In some experiments, arteries were pre-contracted with EC80 U46619, and concentration response to 14, 15 EEZE performed cumulatively.Non peer reviewe
Nitric oxide-dependent vasodilation is compromised in isolated pulmonary arteries from COX knockout mice
Cyclooxygenase (COX) has two isoforms and is essential for prostanoid synthesis. COX-1 is constitutive whilst COX-2 is induced in inflammation. Two COX products, prostacyclin (PGI2) and thromboxane (TxA2), regulate vessel tone; PGI2 mediates vasodilation and platelet inhibition, and TxA2 opposes this. PGI2 therapies are used in pulmonary arterial hypertension (PAH). Endogenous TxA2/PGI2 has been linked to PAH in animal models, but the mechanism and isoform involved is debated. We hypothesized that pulmonary artery (PA) from COX-1ā/ā and COX-2ā/ā mice would have altered vasodilatory function compared with wild-type (WT; C57Bl6) mice. Vasomotor responses to contractile and relaxant agents were measured by myography. PA from all mice responded similarly to contraction by high potassium or the TxA2 mimetic, U46619. Relaxation to PGI2 receptor or PPARĪ²/ agonists was also similar in all PAs. However, COX-1ā/ā and, to a lesser extent, COX-2ā/ā PA had impaired vasodilation to acetylcholine (ACh), which stimulates endothelial nitric oxide (NO) release, and COX-1ā/ā PA also dilated less to sodium nitroprusside (SNP); an NO donor that works on smooth muscle (Fig 1). These data indicate an interaction between COX and NO sensing pathways in pulmonary vessels, and have implications for our understanding of PAH.Non peer reviewedFinal Accepted Versio
Role of the endothelium and COX-1 in prostacyclin generation by whole vessels stimulated with different agonists
Prostacyclin is an important cardioprotective hormone produced by the vascular wall, whose synthesis is dependent on cyclo-oxygenase (COX) enzymes. In healthy vessels the endothelium is thought to be the main site of prostacyclin release (Moncada et al 1977). Two isoforms of COX exist, and we have recently published data demonstrating that it is COX-1 rather than COX-2 that drives the production of prostacyclin in mouse aorta (Kirkby et al 2012). In this study we aimed to extend these observations by investigating what proportion of the COX-1 driven aortic prostacyclin production that comes from the endothelium versus the rest of the vessel wall (smooth muscle layers and adventitia). To do this, we explored how removal of the endothelium would influence the ability of aortic tissue to release prostacyclin in response to a range of agonists that are known to activate the endothelium and the vessel wallNon peer reviewe
Role of COX-1 and COX-2 in the release of prostanoids in murine lung and isolated lung fibroblasts
Cyclooxygenase (COX) is the first enzyme in the conversion of arachidonic acid to prostanoids. There are two isoforms of COX; COX-1, which is constitutively expressed with a homeostatic role in most tissues, and COX-2, which while constitutively expressed in some discreet sites is generally inducible by growth factors and during inflammation. In the current study, we have used tissues and cells from knock-out mice to investigate the relative contributions of COX-1 and COX-2 to PGE2 production by lung tissue ex vivo and by proliferating lung fibroblasts in vitro. Lung tissues from WT (C57Bl6), COX-1-/- and COX-2-/- mice were immediately dissected (<15 min after death) and incubated (37 Ā°C) for 30 min in DMEM containing 50 ĀµM calcium ionophore (A23187). Release of PGE2 was determined by competitive immunoassay. In parallel studies, murine lung fibroblasts from COX-1-/- and COX-2-/- mice were explanted and cultured before being seeded in 96-well plates at sub-confluence (5000-8000/well) and incubated for 24-48 hours in the presence of 10% FCS. Accumulated release of PGE2 was then measured as above. Over 30 min PGE2 was released by lung pieces from wild type (1117 Ā± 55 pg/ml) and COX-2-/- (2013 Ā± 255 pg/ml) but not from COX-1-/- (<61pg/ml) mice (n=4). In contrast, proliferating lung fibroblasts from COX-1-/- (4978.9 Ā± 1392 pg/ml) mice released higher levels of PGE2 than cells from COX-2-/- (1194 Ā± 617 ng/ml) mice (n=4 using cells from 2-3 separate mice for each genotype). These results show that COX-1 activity underpins the stimulated release of PGE2 in healthy mouse lung tissue. Conversely, COX-2 activity predominates in proliferating lung fibroblasts, which may be important as COX-derived PGE2 mediates proliferation of lung fibroblasts (Trends Immunol.2004;25(1):40-6). Our results suggest a switch in COX isoform in lung cells during proliferation which could be relevant to our understanding of conditions such as idiopathic pulmonary fibrosis.Non peer reviewedFinal Accepted Versio
Co-Incubation with PPARĪ²/Ī“ Agonists and Antagonists Modeled Using Computational Chemistry: Effect on LPS Induced Inflammatory Markers in Pulmonary Artery
Ā© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)Peroxisome proliferator activated receptor beta/delta (PPARĪ²/Ī“) is a nuclear receptor ubiquitously expressed in cells, whose signaling controls inflammation. There are large discrepan-cies in understanding the complex role of PPARĪ²/Ī“ in disease, having both antiā and proāeffects on inflammation. After ligand activation, PPARĪ²/Ī“ regulates genes by two different mechanisms; induction and transrepression, the effects of which are difficult to differentiate directly. We studied the PPARĪ²/Ī“āregulation of lipopolysaccharide (LPS) induced inflammation (indicated by release of nitrite and ILā6) of rat pulmonary artery, using different combinations of agonists (GW0742 or Lā165402) and antagonists (GSK3787 or GSK0660). LPS induced release of NO and ILā6 is not significantly reduced by incubation with PPARĪ²/Ī“ ligands (either agonist or antagonist), however, co-incubation with an agonist and antagonist significantly reduces LPSāinduced nitrite production and Nos2 mRNA expression. In contrast, incubation with LPS and PPARĪ²/Ī“ agonists leads to a significant increase in Pdkā4 and Angptlā4 mRNA expression, which is significantly decreased in the presence of PPARĪ²/Ī“ antagonists. Docking using computational chemistry methods indicates that PPARĪ²/Ī“ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. Dual binding in the PPARĪ²/Ī“ binding pocket indicates the ligands retain similar binding energies, which suggests that coāincubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARĪ²/Ī“ binding pocket. To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARĪ²/Ī“ binding pocket has been explored. Agonist binding followed by antagonist simultaneously switches the PPARĪ²/Ī“ mode of action from induction to transrepression, which is linked with an increase in Nos2 mRNA expression and nitrite production.Peer reviewedFinal Published versio
Evidence that Diclofenac and Celecoxib are thyroid hormone receptor beta antagonists
This document is the Accepted Manuscript version, made available under the terms of of the Creative Commons Attribution-NonCommercial-NoDerivatives License CC BY NC-ND 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/ ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.Long term use of NSAIDs is linked to side effects such as gastric bleeding and myocardial infarction. AIMS: Use of in silico methods and pharmacology to investigate the potential for NSAIDs diclofenac, celecoxib and naproxen to bind to nuclear receptors. MATERIALS AND METHODS: In silico screening predicted that both diclofenac and celecoxib has the potential to bind to a number of different nuclear receptors; docking analysis confirmed a theoretical ability for diclofenac and celecoxib but not naproxen to bind to TRĪ². KEY FINDINGS: Results from TRĪ² luciferase reporter assays confirmed that both diclofenac and celecoxib display TRĪ² antagonistic properties; celecoxib, IC50 3.6Ć10(-6)M, and diclofenac IC50 5.3Ć10(-6)M, comparable to the TRĪ² antagonist MLS (IC50 3.1Ć10(-6)M). In contrast naproxen, a cardio-sparing NSAID, lacked TRĪ² antagonist effects. In order to determine the effects of NSAIDs in whole organ in vitro, we used isometric wire myography to measure the changes to Triiodothyronine (T3) induced vasodilation of rat mesenteric arteries. Incubation of arteries in the presence of the TRĪ² antagonist MLS000389544 (10(-5)M), as well as diclofenac (10(-5)M) and celecoxib (10(-5)M) but not naproxen significantly inhibited T3 induced vasodilation compared to controls. SIGNIFICANCE: These results highlight the benefits of computational chemistry methods used to retrospectively analyse well known drugs for side effects. Using in silico and in vitro methods we have shown that both celecoxib and diclofenac but not naproxen exhibit off-target TRĪ² antagonist behaviour, which may be linked to their detrimental side effectsPeer reviewe
The PPARĪ²/Ī“ agonist GW0742 prevents LPS-induced nitrite production in rat parenchyma but not in aorta or pulmonary arteries
Peroxisome Proliferator activated receptors (PPARs) are therapeutic targets in the treatment of inflammatory lung disease. The PPARĪ²/Ī“ agonist GW0742 has potent anti-inflammatory effects in the vasculature (Kapoor et al 2010) which has been linked to a decrease in the production of iNOS in the heart (Kapoor 2010) and activation of Akt-eNOS in arteries (Quintela et al 2014). Here in this study we measured changes in LPS induced NO production in rat arteries and lung parenchyma. Male Wistar (300-350g) rats were killed by CO2 followed by cervical dislocation, and the aorta, conductance and resistance pulmonary arteries and lung parenchyma were dissected under sterile conditions, and placed into 24 well plates. Following incubation with 1Āµg/ml LPS with/without 10-7M GW0742 tissues were incubated for 24 hours, and Griess assay performed to measure nitrite production (a measure of NO release).Our results show that LPS induces a significant increase in NO production from arteries and parenchyma (figure 1). Incubation with GW0742 alone has no effect on basal nitrite levels and does not have an effect on LPS-induced NO production in all types of arteries. In comparison, GW0742 significantly reduces LPS induced NO release in lung parenchyma comparable to inhibition by 10-4M L-NAME and 10-5M 1400W.Figure 1. 2mm rings of aorta, conductance pulmonary artery (CPS), resistance pulmonary artery (RPA) and 1mm2 lung parenchyma strips (lung) were incubated with 1ug/ml LPS Ā± 10-7M GW0742 in DMEM for 24 hours. Supernatant was removed and Griess assay performed to measure nitrite. Data is expressed as mean Ā± SEM; * and f denote p<0.05 by one way ANOVA and Tukeyās post-hoc test, respectively.In summary, incubation for 24 hours with 10-7M GW0742 significantly reduced LPS induced nitrite production in lung parenchyma but not in aorta or pulmonary arteries (conductance and resistance). These data suggest that the effects of PPARĪ²/Ī“ agonists are tissue specific and might support their use as anti-inflammatory agents in lung disease. Kapoor et al Am J Respir Crit Care Med (2010):182; 1506ā1515Quintela et al British Journal of Pharmacology (2014):171; 3089ā3102Peer reviewe
Discovery of novel small molecule inhibitors of S100P, with in vitro anti-metastatic effects on pancreatic cancer cells
Ā© 2020 The Author(s). This is an open access article published under the terms of the Creative Commons Attribution 4.0 International licence (CC BY 4.0). For further details please see https://creativecommons.org/licenses/by/4.0/.S100P, a calcium- binding protein, is known to advance tumor progression and metastasis in pancreatic and several other cancers. Herein is described the in silico identification of a putative binding pocket of S100P to identify, synthesize and evaluate novel small molecules with the potential to selectively bind S100P and inhibit its activation of cell survival and metastatic pathways. The virtual screening of a drug-like database against the S100P model led to the identification of over 100 clusters of diverse scaffolds. A representative test set identified a number of structurally unrelated hits that inhibit S100P-RAGE interaction, measured by ELISA, and reduce in vitro cell invasion selectively in S100P-expressing pancreatic cancer cells at 10 ĀµM. This study establishes a proof of concept in the potential for rational design of small molecule S100P inhibitors for drug candidate development.Peer reviewe
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