Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy

Aims/hypothesis: In previous studies we have shown that extravasated, modified LDL is associated with pericyte loss, an early feature of diabetic retinopathy (DR). Here we sought to determine detailed mechanisms of this LDLinduced pericyte loss.

Methods: Human retinal capillary pericytes (HRCP) were exposed to ‘highly-oxidised glycated’ LDL (HOG-LDL) (a model of extravasated and modified LDL) and to 4-hydroxynonenal or 7-ketocholesterol (components of oxidised LDL), or to native LDL for 1 to 24 h with or without 1 h of pretreatment with inhibitors of the following: (1) the scavenger receptor (polyinosinic acid); (2) oxidative stress (N-acetyl cysteine); (3) endoplasmic reticulum (ER) stress (4-phenyl butyric acid); and (4) mitochondrial dysfunction (cyclosporin A). Oxidative stress, ER stress, mitochondrial dysfunction, apoptosis and autophagy were assessed using techniques including western blotting, immunofluorescence, RT-PCR, flow cytometry and TUNEL assay. To assess the relevance of the results in vivo, immunohistochemistry was used to detect the ER stress chaperon, 78 kDa glucose-regulated protein, and the ER sensor, activating transcription factor 6, in retinas from a mouse model of DR that mimics exposure of the retina to elevated glucose and elevated LDL levels, and in retinas from human participants with and without diabetes and DR.

Results: Compared with native LDL, HOG-LDL activated oxidative and ER stress in HRCP, resulting in mitochondrial dysfunction, apoptosis and autophagy. In a mouse model of diabetes and hyperlipidaemia (vs mouse models of either condition alone), retinal ER stress was enhanced. ER stress was also enhanced in diabetic human retina and correlated with the severity of DR.

Conclusions/interpretation: Cell culture, animal, and human data suggest that oxidative stress and ER stress are induced by modified LDL, and are implicated in pericyte loss in DR.

The use of topical anaesthesia during repair of minor lacerations in Departments of Emergency Medicine:a literature review

There are currently a number of different methods available to obtain anaesthesia in minor dermatological procedures. Although intradermal infiltration of 1% lidocaine is the favoured method for anaesthesia induction in laceration repair, it can cause significant pain in itself. Topical anaesthesia has been investigated as an alternative to infiltration anaesthesia, with the majority of studies looking at preparations of either TAC (tetracaine, adrenaline and cocaine) or LAT (lidocaine, adrenaline and tetracaine).

Native and modified LDL activate extracellular signal-regulated kinases in mesangial cells

Glycation and/or oxidation of LDL may promote diabetic nephropathy. The mitogen-activated protein kinase (MAPK) cascade, which includes extracellular signal-regulated protein kinases (ERKs), modulates cell function. Therefore, we examined the effects of LDL on ERK phosphorylation in cultured rat mesangial cells. In cells exposed to 100 microg/ml native LDL or LDL modified by glycation, and/or mild or marked (copper-mediated) oxidation, ERK activation peaked at 5 min. Five minutes of exposure to 10-100 microg/ml native or modified LDL produced a concentration-dependent (up to sevenfold) increase in ERK activity. Also, 10 microg/ml native LDL and mildly modified LDL (glycated and/or mildly oxidized) produced significantly greater ERK activation than that induced by copper-oxidized LDL +/- glycation (P <0.05). Pretreatment of cells with Src kinase and MAPK kinase inhibitors blocked ERK activation by 50-80% (P <0.05). Native and mildly modified LDL, which are recognized by the native LDL receptor, induced a transient spike of intracellular calcium. Copper-oxidized (+/- glycation) LDL, recognized by the scavenger receptor, induced a sustained rise in intracellular calcium. The intracellular calcium chelator (EGTA/AM) further increased ERK activation by native and mildly modified LDL (P <0.05). These findings demonstrate that native and modified LDL activate ERKs 1 and 2, an early mitogenic signal, in mesangial cells and provide evidence for a potential link between modified LDL and the development of glomerular injury in diabetes.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of <1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase

Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

International differences in sport medicine access and clinical management

I undertook the 2012 ECOSEP travelling fellowship, sponsored by Bauerfeind, between May and August 2012, which involved visiting 5 European sport medicine centres and spending approximately one week in each centre. The 5 centres included: National Track and Field Centre, SEGAS, Thessaloniki, Greece; Professional School in Sport & Exercise Medicine, University of Barcelona, Spain; Sport Medicine Frankfurt Institute, Germany; Isokinetic Medical Group, FIFA Medical Centre of Excellence, Bologna, Italy, and Centre for Sports and Exercise Medicine, Barts and the London School of Medicine and Dentistry, Mile End Hospital, England. Throughout the fellowship, the clinical cases which were routinely encountered were documented. The following sections detail my experiences throughout the fellowship, the sports of the athletes and the injuries which were treated at each of the sport medicine centres during the fellowship visit and the different forms of management employed. © 2012, CIC Edizioni Internazionali

The impact of personalised therapies on respiratory medicine

Stratified approaches to treating disease are very attractive, as efficacy is maximised by identifying responders using a companion diagnostic or by careful phenotyping. This approach will spare non-responders form potential side-effects. This has been pioneered in oncology where single genes or gene signatures indicate tumours that will respond to specific chemotherapies. Stratified approaches to the treatment of asthma with biological therapies are currently being extensively studied. In cystic fibrosis (CF), therapies have been developed that are targeted at specific functional classes of mutations. Ivacaftor, the first of such therapies, potentiates dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein Class III mutations and is now available in the USA and some European countries. Pivotal studies in patients with a G551D mutation, the most common Class III mutation, have demonstrated significant improvements in clinically important outcomes such as spirometry and exacerbations. Sweat chloride was significantly reduced demonstrating a functional effect on the dysfunctional CFTR protein produced by the G551D mutation. Symptom scores are also greatly improved to a level that indicates that this is a transformational treatment for many patients. This stratified approach to the development of therapies based on the functional class of the mutations in CF is likely to lead to new drugs or combinations that will correct the basic defect in many patients with CF. © ERS 2013.

Fortune favours the bold: a higher predator reduces the impact of a native but not an invasive intermediate predator

Emergent multiple predator effects (MPEs) might radically alter predictions of predatory impact that are based solely on the impact of individuals. In the context of biological invasions, determining if and how the individual-level impacts of invasive predators relates to their impacts in multiple-individual situations will inform understanding of how such impacts might propagate through recipient communities. Here, we use functional responses (the relationship between prey consumption rate and prey density) to compare the impacts of the invasive freshwater mysid crustacean Hemimysis anomala with a native counterpart Mysis salemaai when feeding on basal cladoceran prey (i) as individuals, (ii) in conspecific groups and (iii) in conspecific groups in the presence of a higher fish predator, Gasterosteus aculeatus. In the absence of the higher predator, the invader consumed significantly more basal prey than the native, and consumption was additive for both mysid species - that is, group consumption was predictable from individual-level consumption. Invaders and natives were themselves equally susceptible to predation when feeding with the higher fish predator, but an MPE occurred only between the natives and higher predator, where consumption of basal prey was significantly reduced. In contrast, consumption by the invaders and higher predator remained additive. The presence of a higher predator serves to exacerbate the existing difference in individual-level consumption between invasive and native mysids. We attribute the mechanism responsible for the MPE associated with the native to a trait-mediated indirect interaction, and further suggest that the relative indifference to predator threat on the part of the invader contributes to its success and impacts within invaded communities.

Personalized medicine: Has it started yet? A reconstruction of the early history

Within the last few years the field personalized medicine entered the stage. Accompanied with great hopes and expectations it is believed that this field may have the potential to revolutionize medical and clinical care by utilizing genomics information about the individual patients themselves. In this paper, we reconstruct the early footprints of personalized medicine as reflected by information retrieved from PubMed and Google Scholar. That means we are providing a data-driven perspective of this field to estimate its current status and potential problems.