Anthony D. Postle

Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development

Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA2 is a causative agent. Here we show that selective inhibition of Lp-PLA2 with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA2 activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA2 inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke.

Human CD1-restricted T cell recognition of lipids from pollens

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex–related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4+ T cell receptor for antigen (TCR)αβ+, some CD4−CD8− TCRγδ+, but rarely Vα24i + natural killer–T cells, and required CD1a+ and CD1d+ antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-γ, in some cases IL-10 and transforming growth factor-β, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis–exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development

In humans, sexual differentiation of the external genitalia is established at 7-12 weeks post conception (wpc). During this period, maintaining the appropriate intrauterine hormone environment is critical. In contrast to other species, this regulation extends to the human fetal adrenal cortex, as evidenced by the virilization that is associated with various forms of congenital adrenal hyperplasia. The mechanism underlying these clinical findings has remained elusive. Here we show that the human fetal adrenal cortex synthesized cortisol much earlier than previously documented, an effect associated with transient expression of the orphan nuclear receptor nerve growth factor IB-like (NGFI-B) and its regulatory target, the steroidogenic enzyme type 2 3beta-hydroxysteroid dehydrogenase (HSD3B2). This cortisol biosynthesis was maximal at 8-9 wpc under the regulation of ACTH. Negative feedback was apparent at the anterior pituitary corticotrophs. ACTH also stimulated the adrenal gland to secrete androstenedione and testosterone. In concert, these data promote a distinctive mechanism for normal human development whereby cortisol production, determined by transient NGFI-B and HSD3B2 expression, provides feedback at the anterior pituitary to modulate androgen biosynthesis and safeguard normal female sexual differentiation.

Equivalent Effects of Snake PLA2 Neurotoxins and Lysophospholipid-Fatty Acid Mixtures

Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular junction (NMJ). Upon intoxication, the NMJ enlarges and has a reduced content of synaptic vesicles, and primary neuronal cultures show synaptic swelling with surface exposure of the lumenal domain of the synaptic vesicle protein synaptotagmin I. Concomitantly, these neurotoxins induce exocytosis of neurotransmitters. We found that an equimolar mixture of lysophospholipids and fatty acids closely mimics all of the biological effects of SPANs. These results draw attention to the possible role of local lipid changes in synaptic vesicle release and provide new tools for the study of exocytosis.

Application of ’omics technologies to biomarker discovery in inflammatory lung diseases

Inflammatory lung diseases are highly complex in respect of pathogenesis and relationships between inflammation, clinical disease and response to treatment. Sophisticated large-scale analytical methods to quantify gene expression (transcriptomics), proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in the lungs, blood and urine are now available to identify biomarkers that define disease in terms of combined clinical, physiological and patho-biological abnormalities. The aspiration is that these approaches will improve diagnosis, i.e. define pathological phenotypes, and facilitate the monitoring of disease and therapy, and also, unravel underlying molecular pathways. Biomarker studies can either select predefined biomarker(s) measured by specific methods or apply an “unbiased” approach involving detection platforms that are indiscriminate in focus. This article reviews the technologies presently available to study biomarkers of lung disease within the ’omics field. The contributions of the individual ’omics analytical platforms to the field of respiratory diseases are summarised, with the goal of providing background on their respective abilities to contribute to systems medicine-based studies of lung disease. Summary of the application of ’omics-based analytical platforms for biomarker discovery in inflammatory lung diseases http://ow.ly/mjGGc

A comparison of the molecular species compositions of mammalian lung surfactant phospholipids

While dipalmitoyl phosphatidylcholine (PC16:0/16:0) is essential for pulmonary surfactant function, roles for other individual molecular species of surfactant phospholipids have not been established. If any phospholipid species other than PC16:0/16:0 is important for surfactant function, then it may be conserved across animal species. Consequently, we have quantified, by electrospray ionisation mass spectrometry, molecular species compositions of phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) in surfactants from human, rabbit, rat and guinea pig lungs. While PC compositions displayed only relatively minor variations across the animal species studied, there were wide variations of PG and PI concentrations and compositions. Human surfactant PG and PI were enriched in the same three monounsaturated species (PG16:0/18:1, PG18:1/18:1 and PG18:0/18:1) with minimal amounts of PG16:0/16:0 or polyunsaturated species, while all animal surfactant PG contained increased concentrations of PG16:0/16:0 and PG16:0/18:2. Animal surfactant PIs were essentially monounsaturated except for a high content of PI18:0/20:4 (29%) in the rat. As these four surfactants all maintain appropriate lung function of the respective animal species, then all their varied compositions of acidic phospholipids must be adequate at promoting the processes of adsorption, film refinement, respreading and collapse characteristic of surfactant. We conclude that this effectively monounsaturated composition of anionic phospholipid molecular species is a common characteristic of mammalian surfactants.

Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.

Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase α is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ. We report that chromatin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pathway. First, membrane-free nuclei retain all three CDP-choline pathway enzymes in proportions comparable with the content of chromatin-associated phosphatidylcholine. Second, following supplementation of cells with deuterated choline and using electrospray ionization mass spectrometry, both the time course and molecular species labeling pattern of newly synthesized endonuclear and whole cell phosphatidylcholine revealed the operation of spatially separate, compositionally distinct biosynthetic routes. Specifically, endogenous and newly synthesized endonuclear phosphatidylcholine species are both characterized by a high degree of diacyl/alkylacyl chain saturation. This unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depletion and when proliferation is restored, suggesting that endonuclear disaturated phosphatidylcholine enrichment is essential and closely regulated. We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers. Furthermore, our estimates of saturated phosphatidylcholine nuclear volume occupancy of around 10% may imply a significant additional role in regulating chromatin structure.

Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein

There is increasing evidence that modified phospholipid products of low density lipoprotein (LDL) oxidation mediate inflammatory processes within vulnerable atherosclerotic lesions. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is present in vulnerable plaque regions where it acts on phospholipid oxidation products to generate the pro-inflammatory lysophsopholipids and oxidized non-esterified fatty acids. This association together with identification of circulating Lp-PLA2 levels as an independent predictor of cardiovascular disease provides a rationale for development of Lp-PLA2 inhibitors as therapy for atherosclerosis. Here we report a systematic analysis of the effects of in vitro oxidation in the absence and presence of an Lp-PLA2 inhibitor on the phosphatidylcholine (PC) composition of human LDL. Mass spectrometry identifies three classes of PC whose concentration is significantly enhanced during LDL oxidation. Of these, a series of molecules, represented by peaks in the m/z range 594-666 and identified as truncated PC oxidation products by accurate mass measurements using an LTQ Orbitrap mass spectrometer, are the predominant substrates for Lp-PLA2. A second series of oxidation products, represented by peaks in the m/z range 746-830 and identified by LTQ Orbitrap analysis as non-truncated oxidized PCs, are quantitatively more abundant but are less efficient Lp-PLA2 substrates. The major PC products of Lp-PLA2, saturated and mono-unsaturated lyso-PC, constitute the third class. Mass spectrometric analysis confirms the presence of many of these PCs within human atherosclerotic lesions, suggesting that they could potentially be used as in vivo markers of atherosclerotic disease progression and response to Lp-PLA2 inhibitor therapy.

Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila.

Summary Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts.

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-d9-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.