Intimal smooth muscle cells are a source but not a sensor of anti-inflammatory CYP450 derived oxylipins

AbstractVascular pathologies are associated with changes in the presence and expression of morphologically distinct vascular smooth muscle cells. In particular, in complex human vascular lesions and models of disease in pigs and rodents, an intimal smooth muscle cell (iSMC) which exhibits a stable epithelioid or rhomboid phenotype in culture is often found to be present in high numbers, and may represent the reemergence of a distinct developmental vascular smooth muscle cell phenotype. The CYP450-oxylipin - soluble epoxide hydrolase (sEH) pathway is currently of great interest in targeting for cardiovascular disease. sEH inhibitors limit the development of hypertension, diabetes, atherosclerosis and aneurysm formation in animal models. We have investigated the expression of CYP450-oxylipin-sEH pathway enzymes and their metabolites in paired intimal (iSMC) and medial (mSMC) cells isolated from rat aorta. iSMC basally released significantly larger amounts of epoxy-oxylipin CYP450 products from eicosapentaenoic acid > docosahexaenoic acid > arachidonic acid > linoleic acid, and expressed higher levels of CYP2C12, CYP2B1, but not CYP2J mRNA compared to mSMC. When stimulated with the pro-inflammatory TLR4 ligand LPS, epoxy-oxylipin production did not change greatly in iSMC. In contrast, LPS induced epoxy-oxylipin products in mSMC and induced CYP2J4. iSMC and mSMC express sEH which metabolizes primary epoxy-oxylipins to their dihydroxy-counterparts. The sEH inhibitors TPPU or AUDA inhibited LPS-induced NFκB activation and iNOS induction in mSMC, but had no effect on NFκB nuclear localization or inducible nitric oxide synthase in iSMC; effects which were recapitulated in part by addition of authentic epoxy-oxylipins. iSMCs are a rich source but not a sensor of anti-inflammatory epoxy-oxylipins. Complex lesions that contain high levels of iSMCs may be more resistant to the protective effects of sEH inhibitors

An Extensive Literature Review on Neonatal Pain Assessment & Management

Neonatal pain assessment and management is a field requiring much more research. This literature review outlines the current climate of neonatal pain assessment, compares a variety of neonatal pain scales on validity and clinical utility, and implications for how neonatal pain management can be improved. Neonates experience pain to the same degree, if not more, than everyone else. Unmanaged pain during the neonatal period leads to adverse health outcomes. In order to prevent these atrocities from this vulnerable population, NICU pain assessing needs to become the standard of care

A Role for PPARβ/δ in Ocular Angiogenesis

The uses of highly selective PPARβ/δ ligands and PPARβ/δ knockout mice have shown a direct ability of PPARβ/δ to regulate angiogenesis in vitro and in vivo in animal models. PPARβ/δ ligands induce the proangiogenic growth factor VEGF in many cells and tissues, though its actions in the eye are not known. However, virtually, all tissue components of the eye express PPARβ/δ. Both angiogenesis and in particular VEGF are not only critical for the development of the retina, but they are also a central component in many common pathologies of the eye, including diabetic retinopathy and age-related macular degeneration, the most common causes of blindness in the Western world. This review, therefore, will discuss the recent evidence of PPARβ/δ-mediated angiogenesis and VEGF release in the context of ocular disorders

Evidence that MEK1 positively promotes interhomologue double-strand break repair

During meiosis there is an imperative to create sufficient crossovers for homologue segregation. This can be achieved during repair of programmed DNA double-strand breaks (DSBs), which are biased towards using a homologue rather than sister chromatid as a repair template. Various proteins contribute to this bias, one of which is a meiosis specific kinase Mek1. It has been proposed that Mek1 establishes the bias by creating a barrier to sister chromatid repair, as distinct from enforcing strand invasion with the homologue. We looked for evidence that Mek1 positively stimulates strand invasion of the homologue. This was done by analysing repair of DSBs induced by the VMA1- derived endonuclease (VDE) and flanked by directly repeated sequences that can be used for intrachromatid single-strand annealing (SSA). SSA competes with interhomologue strand inva- sion significantly more successfully when Mek1 function is lost. We suggest the increase in intrachromosomal SSA reflects an opportunistic default repair pathway due to loss of a MEK1 stimulated bias for strand invasion of the homologous chromosome. Making use of an inhibitor sensitive mek1-as1 allele, we found that Mek1 function influences the repair pathway throughout the first 4-5 h of meiosis. Perhaps reflecting a particular need to create bias for successful interhomologue events before chromosome pairing is complete. © The Author(s) 2010. Published by Oxford University Pres

The Role of PPARs in the Endothelium: Implications for Cancer Therapy

The growth and metastasis of cancers intimately involve the vasculature and in particular the endothelial cell layer. Tumours require new blood vessel formation via angiogenesis to support growth. In addition, inflammation, coagulation, and platelet activation are common signals in the growth and metastasis of tumour cells. The endothelium plays a central role in the homeostatic control of inflammatory cell recruitment, regulating platelet activation and coagulation pathways. PPARα, -β/δ, and -γ are all expressed in endothelial cells. This review will discuss the roles of PPARs in endothelial cells in relation to angiogenesis, inflammation, coagulation, and platelet control pathways. In particular, we will discuss the recent evidence that supports the hypothesis that PPARα and PPARγ are antiangiogenic receptors, while PPARβ/δ is proangiogenic

Time trials versus time to exhaustion tests: Effects on critical 1 power, W′ and oxygen uptake kinetics

Purpose: To investigate single-day time-to-exhaustion (TTE) and time trial (TT) based laboratory tests values of critical power (CP), Wprime (W') and respective oxygen kinetics responses. Methods: Twelve cyclists performed a maximal ramp test followed by three TTE and three TT efforts interspersed by a 60-min recovery between efforts. Oxygen uptake was measured during all trials. The mean response time (MRT) was calculated as a description of the overall V ̇O2 kinetic response from the onset to 2 min of exercise. Results: TTE determined CP was 279 ± 52W and TT determined CP was 276 ± 50W (P = 0.237). Values of W were 14.3 ± 3.4 kJ (TTE W') and 16.5± 4.2 kJ (TT W') (P = 0.028). Whilst a high level of agreement (-12 to 17 W) and a low prediction error of 2.7% was established for CP, for W limits of agreements were markedly lower (-8 to 3.7 kJ) with a prediction error of 18.8%. The mean standard error for TTE CP values was significantly higher than that for TT CP values (2.4 ± 1.9% vs. 1.2 ± 0.7% W). The standard error for TTE W and TT W were 11.2 ± 8.1% and 5.6 ± 3.6%, respectively. The V ̇O2 response was significantly faster during TT (~22 s) than TTE (~28 s). Conclusions: The time-trial protocol with a 60-min recovery period offers a valid, time-saving and less error containing alternative to conventional and more recent testing methods. Results however cannot be transferred to W'

Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity

Pregnane X receptor regulates drug metabolism and transport in the vasculature and protects from oxidative stress

Aims Circulating endogenous, dietary and foreign chemicals can contribute to vascular dysfunction. The mechanism by which the vasculature protects itself from these chemicals is unknown. This study investigates whether the pregnane X receptor (PXR), the major transcriptional regulator of hepatic drug metabolism and transport that responds to such xenobiotics, mediates vascular protection by co-ordinating a defence gene program in the vasculature.Methods and Results PXR was detected in primary human and rat aortic endothelial and smooth muscle cells and blood vessels including human and rat aorta. Metabolic PXR target genes cytochrome P450 3A, 2B, 2C and glutathione-S-transferase mRNA and activity were induced by PXR ligands in rodent and human vascular cells and absent in the aortas from PXR null mice stimulated in vivo or in rat aortic smooth muscle cells expressing dominant negative PXR. Activation of aortic PXR by classical agonists had several protective effects; increased xenobiotic metabolism demonstrated by bio-activation of the pro-drug clopidogrel, which reduced adenosine diphosphate-induced platelet aggregation; increased expression of multidrug resistance protein 1, mediating chemical efflux from the vasculature; and protection from reactive oxygen species-mediated cell death.Conclusions PXR co-ordinately up-regulates drug metabolism, transport and anti-oxidant genes to protect the vasculature from endogenous and exogenous insults, thus representing a novel gatekeeper for vascular defence

Computational modelling of the binding of arachidonic acid to the human monooxygenase CYP2J2

An experimentally determined structure for human CYP2J2—a member of the cytochrome P450 family with significant and diverse roles across a number of tissues—does not yet exist. Our understanding of how CYP2J2 accommodates its cognate substrates and how it might be inhibited by other ligands thus relies on our ability to computationally predict such interactions using modelling techniques. In this study we present a computational investigation of the binding of arachidonic acid (AA) to CYP2J2 using homology modelling, induced fit docking (IFD) and molecular dynamics (MD) simulations. Our study reveals a catalytically competent binding mode for AA that is distinct from a recently published study that followed a different computational pipeline. Our proposed binding mode for AA is supported by crystal structures of complexes of related enzymes to inhibitors, and evolutionary conservation of a residue whose role appears essential for placing AA in the right site for catalysis