Megan Kaneda

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing.

Renal vascular inflammation induced by Western diet in ApoE-null mice quantified by 19F NMR of VCAM-1 targeted nanobeacons

UNLABELLED We have designed multifunctional nanoparticulate reporter bioprobes capable of targeting vascular cell adhesion molecule 1 (VCAM-1), which is up-regulated in numerous inflammatory processes. These perfluorocarbon-cored nanoparticles emit a unique (19)F magnetic resonance (MR) signature, providing the potential to localize and quantify VCAM-1 expression in early atherosclerosis. Nanoparticle-VCAM-1 targeting specificity was confirmed by in vitro binding and competition studies. ApoE-null and control C57-BL6 mice (n = 6/group), fed a Western diet for 35 weeks, were injected i.v. with targeted or non-targeted nanoparticles. After two hours, kidneys were excised and prepared for analysis. ApoE-null kidneys exhibited increased VCAM-1-targeted nanoparticle content over healthy controls by (19)F MR spectroscopy (36.5+8.8 vs. 9.3+2.2 x 10(8)/g, P < .05), which correlated with increased VCAM-1 staining (2.5 +/- 1.3% vs. 0.9 +/- 0.3%, P < .05); their relative biodistributions were confirmed by fluorescence microscopy and MR imaging. These molecular imaging agents offer new approaches for detection, quantification, and longitudinal evaluation of early inflammation utilising (19)F MR spectroscopy and imaging. FROM THE CLINICAL EDITOR Multifunctional nanoparticulate reporter bioprobes capable of targeting vascular cell adhesion molecule 1 (VCAM-1) are reported in this paper. These perfluorocarbon-cored nanoparticles offer new approaches for detection, quantification, and longitudinal evaluation of early inflammation utilising 19F MR spectroscopy and imaging.

Mechanisms of nucleotide trafficking during siRNA delivery to endothelial cells using perfluorocarbon nanoemulsions.

RNA interference (RNAi) is a useful in vitro research tool, but its application as a safe and effective therapeutic agent may benefit from improved understanding of mechanisms of exogenous siRNA delivery, including cell trafficking and sorting patterns. We report the development of a transfection reagent for siRNA delivery which employs a distinctive non-digestive mode of particle-cell membrane interaction through the formation of a hemifusion complex resulting in lipid raft transport of cargo to the cytosol, bypassing the usual endosomal nanoparticle uptake pathway. We further demonstrate markedly enhanced efficacy over conventional transfection agents for suppressing endothelial cell expression of upregulated vascular adhesion molecules.

Fluid Shear Stress Modulates Cell Migration Induced by Sphingosine 1-Phosphate and Vascular Endothelial Growth Factor

The rational design of drug delivery systems requires the ability to predict the environment-specific responses of target cells to the delivered drug. Here we describe the in vitro effects of fluid shear stress, vascular endothelial growth factor (VEGF), and sphingosine 1-phosphate (S1P) on the migration of human umbilical vein endothelial cells (HUVEC). Endothelial cell migration into a scrape wound was enhanced in S1P- or VEGF-stimulated HUVEC by the addition of fluid shear stress. In both cases, scrape wound closure rates were near a maximal value that was not exceeded when cells were exposed to all three factors. We also found that cell migration into a scrape wound due to S1P stimulation was correlated with the S1P1 mRNA concentration, in systems where cell migration was not already near maximal. The present work represents our initial steps toward predicting cell migration based upon the activation state of the receptors and enzymes involved in the chemokinetic response. These results also illustrate the importance of context-dependent analysis of cell signaling cascades.

Endothelial cell migration in human plasma is enhanced by a narrow range of added sphingosine 1-phosphate: Implications for biomaterials design

Sphingosine 1-phosphate (S1P) promotes endothelial cell migration in vitro and may potentially impact the endothelialization of implanted biomaterials. However, the effects of S1P on endothelial cells (EC) in flowing blood could be negligible due to preactivation of signaling cascades. We previously developed biomaterials that release S1P and wished to determine through in vitro experiments the extent to which EC respond to S1P added to human platelet poor plasma. We found that addition of 200 nM S1P to platelet poor plasma significantly increased cell migration in two migration models. A lower concentration of S1P added to plasma (100 nM) did not increase endothelial cell migration rates, while the cell migration response was saturated above 200 nM S1P. Expression of the main S1P receptor in EC, S1P(1), was elevated in plasma compared to low serum medium, but addition of VEGF or fluid flow elicited a further increase in S1P(1) mRNA, consistent with the synergistic effects observed between S1P, VEGF, and fluid flow. Thus, sustained delivery of S1P from biomaterials might only enhance endothelial cell migration if the concentration of S1P at the surface of the material stimulated adjacent EC to the same extent as approximately 200 nM S1P added to plasma.

19F MRS assessment of siRNA delivery to vascular cells via perfluorocarbon nanoparticles

Methods To design a synthetic vehicle to serve as both an siRNA delivery agent and an imaging agent, perfluorocarbon nanoparticles (PFC-NP) were loaded with the cationic lipid 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) in the lipid monolayer to form transfection complexes with siRNA to the VCAM-1 gene. siRNA loading onto nanoparticles was measured via PAGE gel. Mouse 2F2B endothelial cells were incubated with transfection complexes (PFC-NP/siRNA) for 4 h. 19F MR spectroscopy was performed at 11.7 T to determine the number of NP bound to each cell. mRNA levels were measured 48 h after transfection to determine knockdown.