Cholesterol Efflux-Independent Modification of Lipid Rafts by AIBP (Apolipoprotein A-I Binding Protein).

OBJECTIVE: AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux. CONCLUSIONS: Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.

The mode of anesthesia influences outcome in mouse models of arterial thrombosis.

BACKGROUND: Arterial thrombosis models are important for preclinical evaluation of antithrombotics but how anesthetic protocol can influence experimental results is not studied. OBJECTIVES: We studied how three most commonly used rodent anesthetics affect the induction of thrombosis and thrombus resolution with antiplatelet agent integrilin (Eptifibatide). METHODS: The Folts, electrolytic, and FeCl3 models of carotid artery thrombosis were evaluated. The extent of blood flow reduction required to elicit cyclic flow reductions (CFR) was examined in the Folts model. The occlusion time and stability following electrolytic or FeCl3 injury was assessed. The efficacy of Eptifibatide was studied in each cohort and clot composition following FeCl3 application was assessed histologically. RESULTS: Isoflurane and ketamine-xylazine (ket-x) elicited higher basal blood flow velocities. For reliable CFR in the Folts model, a higher degree of blood flow reduction was required under ket-x and isoflurane. For the FeCl3 and electrolytic models, injury severity had to be increased in mice under ket-x anesthesia to achieve rapid occlusion. FeCl3-injured artery sections from ket-x and isoflurane-treated mice showed vessel dilatation and clots that were more fibrin/red-cell rich compared to pentobarbitone. Integrilin led to cycle abolishment for all three Folts-injury cohorts but for the electrolytic model a 2.5-fold higher dose was required to restore blood flow under pentobarbitone. Integrilin after FeCl3 arterial injury was partially ineffective in isoflurane-treated mice. CONCLUSIONS: Anesthesia impacts rodent carotid artery occlusion experiments and alters integrilin efficacy. It is important to consider anesthetic protocols in animal experiments involving pharmacological agents for treatment of atherothrombosis.

Low-Fouling and Biodegradable Protein-Based Particles for Thrombus Imaging.

Nanomedicine holds great promise for vascular disease diagnosis and specific therapy, yet rapid sequestration by the mononuclear phagocytic system limits the efficacy of particle-based agents. The use of low-fouling polymers, such as poly(ethylene glycol), efficiently reduces this immune recognition, but these nondegradable polymers can accumulate in the human body and may cause adverse effects after prolonged use. Thus, new particle formulations combining stealth, low immunogenicity and biocompatible features are required to enable clinical use. Here, a low-fouling particle platform is described using exclusively protein material. A recombinant protein with superior hydrophilic characteristics provided by the amino acid repeat proline, alanine, and serine (PAS) is designed and cross-linked into particles with lysine (K) and polyglutamic acid (E) using mesoporous silica templating. The obtained PASKE particles have low-fouling behavior, have a prolonged circulation time compared to albumin-based particles, and are rapidly degraded in the cell's lysosomal compartment. When labeled with near-infrared fluorescent molecules and functionalized with an anti-glycoprotein IIb/IIIa single-chain antibody targeting activated platelets, the particles show potential as a noninvasive molecular imaging tool in a mouse model of carotid artery thrombosis. The PASKE particles constitute a promising biodegradable and versatile platform for molecular imaging of vascular diseases.

Nucleocytoplasmic coagulation: an injury-induced aggregation event that disulfide crosslinks proteins and facilitates their removal by plasmin.

Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seen in human brain tissue following neurotrauma, act as a cofactor and substrate for the plasminogen-activating system. In plasminogen(-/-) mice, levels of misfolded ß-tubulin were elevated and its clearance delayed following neurotrauma, demonstrating a requirement for plasminogen in the removal of NCC constituents. While additional in vivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury.

Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells.

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.

The Netrin receptor Neogenin is required for neural tube formation and somitogenesis in zebrafish.

The Netrin receptor Deleted in colon cancer (Dcc) has been shown to play a pivotal role in the guidance of nascent axons towards the ventral midline in the developing nervous systems of both vertebrates and invertebrates. In contrast, the function during embryogenesis of a second Dcc-like Netrin receptor Neogenin has not yet been defined. We used antisense morpholino oligonucleotides to knockdown Neogenin activity in zebrafish embryos and demonstrate that Neogenin plays an important role in neural tube formation and somitogenesis. In Neogenin knockdown embryos, cavitation within the neural rod failed to occur, producing a neural tube lacking a lumen. Somite formation was also defective, implicating Neogenin in the migration events underlying convergent extension during gastrulation. These observations suggest a role for Neogenin in determining cell polarity or migrational directionality of both neuroectodermal and mesodermal cells during early embryonic development.

The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish.

The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kilobase promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germ line transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)pA301 transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation.