Synthesis and activity of a novel Autotaxin inhibitor-Icodextrin conjugate

© Copyright 2018 American Chemical Society. Autotaxin is an extracellular phospholipase D that catalyses the hydrolysis of lysophosphatidyl choline (LPC) to generate the bioactive lipid lysophosphatidic acid (LPA). Autotaxin has been implicated in many pathological processes relevant to cancer. Intraperitoneal administration of an autotaxin inhibitor may benefit patients with ovarian cancer, however low molecular mass compounds are known to be rapidly cleared from the peritoneal cavity. Icodextrin is a polymer that is already in clinical use because it is slowly eliminated from the peritoneal cavity. Herein we report conjugation of the autotaxin inhibitor HA-155 to icodextrin. The conjugate inhibits autotaxin activity (IC50 = 0.86 ± 0.13 μg mL-1) and reduces cell migration. Conjugation of the inhibitor increased its solubility, decreased its membrane permeability and improved its intraperitoneal retention in mice. These observations demonstrate the first application of icodextrin as a covalently-bonded drug delivery platform with potential use in the treatment of ovarian cancer

Modeling of hydraulic fracture branching in porous media using hybrid FEM coupled with peridynamics

Simulation of complex fracture patterns can aid understanding of the mechanisms of hydraulic fracturing, with potential for significant impact on improving oil and gas recovery. In this paper, a fully coupled hydraulic fracture propagation simulation method employing a hybrid finite element method (FEM) coupled with peridynamics (PD) is presented. Considering the ability of PD for solving discontinuous problems, the solid deformation and fracture propagation are captured by peridynamics (PD) and fluid flow is controlled by FEM. The whole process is solved by a fully implicit method. This paper is addressed to researchers who would like to have a quick and simple numerical implementation of a simulator for computing complex hydraulic fracturing processes. The presented method demonstrates the capability of the model for handling complex dynamic crack propagation together with crack branching. The study shows that at faster loading rate, higher-energy release rate, more brittle and impermeable media will induce crack branching more easily

Phenotype of LSEC from normal and DIO mice.

<p>A) Primary LSEC (CD45^-CD146⁺) isolated from normal C57Bl6 lean mice were analyzed for CD146 and CD31 expression and uptake of DiI-labeled Ac-LDL by flow cytometry. (B) Primary LSEC isolated from C57Bl6 lean mice were cultured with the indicated FFA for 24hours. CCL2 levels in the supernatant were measured by ELISA. Graphs represent the mean +/- SE from 3 mice. Primary LSEC (CD45^-CD146⁺) (C) and LSEC depleted non-parenchymal cells (CD45⁺CD146^-) (D) were isolated from mice fed a low fat diet (NCD) and obese mice fed a high fat diet (HFD) for 12 weeks and gene expression for chemokines was evaluated by quantitative qPCR. Graphs represent the mean +/- SE from 5–7 mice. *p<0.05.</p

Downregulation of chemokines by LSEC in response to treatment with FFA.

<p>TSEC (A) and AML12 (B) were cultured with 0.33mM OA and/or 0.33mM PA for 16 hours. RNA was isolated from cells and levels of the indicated chemokine were determined by quantitative RT PCR. (C) TSEC were treated with 0.33mM of the indicated FFA for 16 hours and CCL2 expression was determined by quantitative RT PCR. (D) TSEC and AML12 were treated with 0.33mM OA and 0.33mM PA for 16 hours followed by 6 hour stimulation with 100ng/ml LPS. CCL2 gene expression (top) and protein production (bottom) were measured. (E) The human LSEC (TMNK) and hepatocyte (HepG2) cell lines were stimulated with FFA and LPS as in (D) and CCL2 gene expression was measured by quantitative PCR. Plots represent the mean +/- SE of three experiments. *p<0.05.</p

KEGG pathways in FFA treated TSEC.

<p>Relevant enriched pathways and the involved genes were determined by analysis of all genes with a p value <0.05 and a fold-change > 1.25 within the KEGG pathway database. </p

Free Fatty Acids Differentially Downregulate Chemokines in Liver Sinusoidal Endothelial Cells: Insights into Non-Alcoholic Fatty Liver Disease

<div><p>Non-alcoholic fatty liver disease is a prevalent problem throughout the western world. Liver sinusoidal endothelial cells (LSEC) have been shown to play important roles in liver injury and repair, but their role in the underlying pathogenetic mechanisms of non-alcoholic fatty liver disease remains undefined. Here, we evaluated the effects of steatosis on LSEC gene expression in a murine model of non-alcoholic fatty liver disease and an immortalized LSEC line. Using microarray we identified distinct gene expression profiles following exposure to free fatty acids. Gene pathway analysis showed a number of differentially expressed genes including those involved in lipid metabolism and signaling and inflammation. Interestingly, in contrast to hepatocytes, fatty acids led to decreased expression of pro-inflammatory chemokines including CCL2 (MCP-1), CXCL10 and CXCL16 in both primary and LSEC cell lines. Chemokine downregulation translated into a significant inhibition of monocyte migration and LSECs isolated from steatotic livers demonstrated a similar shift towards an anti-inflammatory phenotype. Overall, these pathways may represent a compensatory mechanism to reverse the liver damage associated with non-alcoholic fatty liver disease.</p></div

FFA inhibits the migration of monocytes in vitro.

<p>Murine monocytes were isolated from the bone marrow of C56Bl6/J mice and co-cultured for 2 hours in transwell plates with TSEC (A) or AML12 (B) either resting or pre-treated with FFA and LPS for 18 hours. Cells were stained with the monocyte/macrophage markers CD11b, F/480 and Ly6C and the chemotactic index was calculated (#migrated cells in treatment wells/# migrated cell in the control well). (C) Representative histograms showing Ly6C expression on migrated cells. (D) The chemotactic index of Ly6C high and Ly6C low cells towards resting or FFA-treated TSEC. Plots represent the mean +/- SE of three experiments. *p<0.05.</p

Gene expression analysis of TSEC treated with FFA.

<p>TSECs were treated with 0.33mM OA and 0.33mM PA for 16 hours. RNA was isolated from cells and microarray analysis of triplicate wells was performed using IPA (Qiagen). (A) Heat map of the top 20 up-regulated and down-regulated genes in the FFA treated LSEC compared to untreated cells. Individual samples are given on the rows, while genes are listed by column. Gene expression is normalized to the mean and color-scaled by standard deviations above (red) and below (green) the average expression in all six independent samples. (B) Upregulation of genes involved in lipid metabolism genes (red) and the predicted activated pathways (orange) in TSEC treated with FFA. (C) Predicted activation of PPAR<b>α</b> based on Ingenuity upstream regulator analysis. Upregulated genes downstream of PPAR<b>α</b> are shown (red).</p

Suppression of ELAVL-1/HUR leads to down regulation of GSK3β <i>in vitro</i> and subsequently increased of epithelial albumin clearance.

<p><b>(</b>A) ELAVL-1/HUR protein expression and suppression in nuclear cell lysate in cells treated with ELAVL-1/HUR siRNA. (B) mRNA and protein expression of GSK3β in cells transfected with siELLAVL-1. Binding (C) and uptake (D) of FITC labelled albumin All experiments were conducted in A549 cells, n 3–4; data represent the mean ± SEM * p< 0.05, ***p<0.001, ****p<0.0001.</p

Detrimental ELAVL-1/HuR-dependent GSK3β mRNA stabilization impairs resolution in acute respiratory distress syndrome

<div><p>A hallmark of acute respiratory distress syndrome (ARDS) is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK) 3β during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3β could play a functional role in ARDS resolution. To address this hypothesis, we performed an <i>in silico</i> analysis to identify regulatory genes whose expression correlation to GSK3β messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3β, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like) as a central component in a likely GSK3β signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3β mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3β at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3β. Together, our findings indicate a previously unknown interaction between GSK3β and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3β pathways as a novel ARDS treatment approach.</p></div