Alexander Vezeridis

A2b Adenosine Receptor Regulates Hyperlipidemia and Atherosclerosis

Background— The cAMP-elevating A2b adenosine receptor (A2bAR) controls inflammation via its expression in bone marrow cells. Methods and Results— Atherosclerosis induced by a high-fat diet in apolipoprotein E–deficient mice was more pronounced in the absence of the A2bAR. Bone marrow transplantation experiments indicated that A2bAR bone marrow cell signals alone were not sufficient to elicit this effect. Intriguingly, liver expression of the A2bAR in wild-type mice was vastly augmented by a high-fat diet, raising the possibility that this upregulation is of functional significance. A2bAR genetic ablation led to elevated levels of liver and plasma cholesterol and triglycerides and to fatty liver pathology typical of steatosis, assessed by enzymatic assays and analysis of liver sections. Western blotting and quantitative polymerase chain reaction revealed elevated expression of the following molecules in the liver of A2bAR-null mice: the transcription factor sterol regulatory element binding protein-1 (SREBP-1) and its 2 downstream targets and regulators of lipogenesis, acetyl CoA carboxylase and fatty acid synthase. Pharmacological activation or inhibition of A2bAR in primary hepatocytes confirmed the regulation of SREBP-1 by this receptor. A2bAR-mediated changes in cAMP were found to regulate levels of the transcriptionally active form of SREBP-1. Finally, adenovirally mediated restoration of the A2bAR in the liver of A2bAR-null mice reduced the lipid profile and atherosclerosis. Similarly, in vivo administration of the A2bAR ligand BAY 60-6853 in control mice on a high-fat diet reduced the lipid profile and atherosclerosis. Conclusion— This study provides the first evidence that the A2bAR regulates liver SREBP-1, hyperlipidemia, and atherosclerosis, suggesting that this receptor may be an effective therapeutic target.

Discrete roles of apoA-I and apoE in the biogenesis of HDL species: lessons learned from gene transfer studies in different mouse models.

Using adenovirus‐mediated gene transfer in apolipoprotein A‐I (apoA‐I)‐deficient mice, we have established that apoA‐I mutations inhibit discrete steps in a pathway that leads to the biogenesis and remodeling of high‐density lipoprotein (HDL). To this point, five discrete categories of apoA‐I mutants have been characterized that may affect the interactions of apoA‐I with ATP‐binding cassette superfamily A, member 1 (ABCA1) or lecithin:cholesterol acyl transferase (LCAT) or may influence the plasma phospholipid transfer protein activity or may cause various forms of dyslipidemia. Biogenesis of HDL is not a unique property of apoA‐I. Using adenovirus‐mediated gene transfer of apoE in apoA‐I‐ or ABCA1‐deficient mice, we have established that apolipoprotein E (apoE) also participates in a novel pathway of biogenesis of apoE‐containing HDL particles. This process requires the functions of the ABCA1 lipid transporter and LCAT, and it is promoted by substitution of hydrophobic residues in the 261 to 269 region of apoE by Ala. The apoE‐containing HDL particles formed in the circulation may have atheroprotective properties. ApoE‐containing HDL may also have important biological functions in the brain that confer protection from Alzheimers disease.

Contrast Enhanced Ultrasound (CEUS) Liver Imaging Reporting and Data System (LI-RADS®): the official version by the American College of Radiology (ACR)

Author(s): Kono, Yuko; Lyshchik, Andrej; Cosgrove, David; Dietrich, Christoph F; Jang, Hyun-Jung; Kim, Tae Kyoung; Piscaglia, Fabio; Willmann, Juergen K; Wilson, Stephanie R; Santillan, Cynthia; Kambadakone, Avinash; Mitchell, Donald; Vezeridis, Alexander; Sirlin, Claude B

Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia.

Background Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136–150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential. Methodology/Principal Findings In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent. Conclusions/Significance Overall, our findings suggest that single amino acid changes in the functionally important region 136–150 of apoE3 can affect the molecules stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis.

Domains of apoE4 required for the biogenesis of apoE-containing HDL.

Abstract Introduction. We have studied the functions of truncated apoE4 forms in vitro and in vivo in order to identify the domains of apoE4 required for the biogenesis of apoE-containing high-density lipoprotein (HDL). Results. We have found that apoE4-185, -202, -229, or -259 could promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux in vitro, although less efficiently than Full-length apoE4, and had diminished capacity to activate lecithin cholesterol acyltransferase (LCAT). Formation of HDL in vivo was assessed by various methods following gene transfer in apolipoprotein A-I−/− × apoE−/− mice. Fast protein liquid chromatography of plasma showed that the truncated apoE forms, except apoE4-185, generated an apoE-containing HDL peak. Two-dimensional gel electrophoresis of plasma and electron microscopy showed that truncated apoE forms generated distinct HDL subpopulations and formed discoidal HDL particles which could be converted to spherical by co-administration of truncated apoE4-202 and LCAT. Conclusion. Overall, the in-vivo and in-vitro data are consistent and indicate that apoE4-185 is the shortest truncated form that supports formation of discoidal apoE4-containing HDL particles.

Polymer-stabilized perfluorobutane nanodroplets for ultrasound imaging agents

In this paper, we describe a method for the stabilization of low-boiling point (low-bp) perfluorocarbons (PFCs) at physiological temperatures by an amphiphilic triblock copolymer which can emulsify PFCs and be cross-linked. After UV-induced thiol-ene cross-linking, the core of the PFC emulsion remains in liquid form even at temperatures exceeding their boiling points. Critically, the formulation permits vaporization at rarefactional pressures relevant for clinical ultrasound.

Evaluation of Knee Donor and Elbow Recipient Sites for Osteochondral Autologous Transplantation Surgery in Capitellar Osteochondritis Dissecans

Background: Osteochondral autologous transplantation surgery (OATS) has been advocated for treatment of osteochondritis dissecans (OCD) of the capitellum in adolescents. However, little information is available regarding the optimal knee harvest site to match the contour and cartilage thickness of the recipient elbow lesion. Purpose: To characterize the capitellar anatomic structure in adolescents with and without OCD and to compare these measurements to normal adolescent knees to identify the optimal site for osteochondral graft harvest. Study Design: Controlled laboratory study. Methods: Twenty-one patients with OCD were analyzed. Twenty-two patients with normal elbows and 25 age-, weight-, and height-matched patients with normal knees were also identified. Cartilage radii of curvatures (ROCs) in the sagittal and coronal-axial planes were measured on magnetic resonance imaging (MRI) of normal capitella and 5 sites (posterior lateral femoral condyle, medial and lateral middle trochlear ridges, and medial and lateral inferior trochlear ridges) in normal knees. Differences in ROC between the knee donor and capitellar recipient sites were calculated based on a 10-mm osteochondral plug diameter. Results: Overall, the mean apex differences between graft and recipient sites ranged from 0.4 to 0.9 mm, and mean edge differences ranged from 0.5 to 1.4 mm in the coronal-axial dimension. Of all knee sites tested, the posterior lateral femoral condyle had average ROCs (19.1 mm sagittal; 14.1 mm axial) most like the capitellum (10.6 mm sagittal, 12.6 mm coronal-axial), resulting in minimal apex and edge differences (apex difference = −0.6 mm; coronal-axial side difference = −0.5 mm; no sagittal side difference). Of the anterior nonweightbearing sites, the inferior medial trochlear ridge (28.3 mm sagittal ROC; 13.2 mm coronal-axial ROC) demonstrated the lowest apex and side differences when compared with the capitellum (apex difference = −0.8 mm; coronal-axial side difference = −0.8 mm; no sagittal side difference). The frequently used middle lateral trochlear ridge (28.8 mm sagittal; 8.7 mm coronal-axial ROCs) had the largest side difference (apex distance = −0.8 mm; coronal-axial side difference = −1.4 mm; no sagittal side difference). Conclusion/Clinical Relevance: In cases where a large single-plug OATS is considered, a 10-mm plug from the anterior nonweightbearing aspect of the distal femur is calculated to result in ≤1 mm of articular incongruity at the recipient capitellum. The inferior medial trochlear ridge should be considered as a donor site for OATS procedures for OCD given its accessibility and favorable geometry.

Molecular etiology of a dominant form of type III hyperlipoproteinemia caused by R142C substitution in apoE4

We have used adenovirus-mediated gene transfer in apolipoprotein (apo)E−/− mice to elucidate the molecular etiology of a dominant form of type III hyperlipoproteinemia (HLP) caused by the R142C substitution in apoE4. It was found that low doses of adenovirus expressing apoE4 cleared cholesterol, whereas comparable doses of apoE4[R142C] greatly increased plasma cholesterol, triglyceride, and apoE levels, caused accumulation of apoE in VLDL/IDL/LDL region, and promoted the formation of discoidal HDL. Co-expression of apoE4[R142C] with lecithin cholesterol acyltransferase (LCAT) or lipoprotein lipase (LPL) in apoE−/− mice partially corrected the apoE4[R142C]-induced dyslipidemia. High doses of C-terminally truncated apoE4[R142C]-202 partially cleared cholesterol in apoE−/− mice and promoted formation of discoidal HDL. The findings establish that apoE4[R142C] causes accumulation of apoE in VLDL/IDL/LDL region and affects in vivo the activity of LCAT and LPL, the maturation of HDL, and the clearance of triglyceride-rich lipoproteins. The prevention of apoE4[R142C]-induced dyslipidemia by deletion of the 203-299 residues suggests that, in the full-length protein, the R142C substitution may have altered the conformation of apoE bound to VLDL/IDL/LDL in ways that prevent triglyceride hydrolysis, cholesterol esterification, and receptor-mediated clearance in vivo.

Contrast-enhanced ultrasound (CEUS) liver imaging reporting and data system (LI-RADS) 2017 - a review of important differences compared to the CT/MRI system.

Medical imaging plays an important role in the diagnosis and management of hepatocellular carcinoma (HCC). The Liver Imaging Reporting and Data System (LI-RADS) was initially created to standardize the reporting and data collection of CT and MR imaging for patients at risk for HCC. As contrast-enhanced ultrasound (CEUS) has been widely used in clinical practice, it has recently been added to the LI-RADS. While CEUS LI-RADS shares fundamental concepts with CT/MRI LI-RADS, there are key differences between the modalities reflecting dissimilarities in the underlying methods of image acquisition and types of contrast material. This review introduces a recent update of CEUS LI-RADS and explains the key differences from CT/MRI LI-RADS.

In Vivo Transfection and Detection of Gene Expression of Stem Cells Preloaded with DNA-carrying Microbubbles

PURPOSE To determine whether (a) stem cells loaded with DNA-carrying microbubbles (MBs) can be transfected in vivo, (b) the cells remain alive to express the gene, and (c) gene expression is sufficiently robust to be detected in vivo. MATERIALS AND METHODS The study was approved by the Institutional Animal Care and Use Committee. Cationic MBs were prepared, characterized, and loaded with pLuciferase green fluorescent protein (GFP) plasmid. Loading was confirmed with SYBR Gold staining (Life Technologies, Carlsbad, Calif). C17.2 cells were loaded with the DNA-carrying MBs. Two hundred thousand cells suspended in 20 μL phosphate-buffered saline were mixed with 200 μL Matrigel (BD Biosciences, San Jose, Calif) and injected in both flanks of eight nude mice. One of the Matrigel (BD Biosciences) injections contained 50 000 cells pretransfected in vitro by using lipofectamine as a positive control. Nine flanks were exposed to 2.25-MHz ultrasonic pulses at 50% duty cycle for 1 minute at 1 W/cm(2) (n = 3) or 2 W/cm(2) (n = 6), and six flanks served as the negative control. Two days later, bioluminescent images were acquired in each mouse every 3 minutes for 1 hour after the intraperitoneal injection of d-luciferin (Perkin Elmer, Waltham, Mass). Differences between groups were assessed by using the nonparametric Kruskal-Wallis test with Wilcoxon rank sum tests for follow-up comparisons. Mice were then killed, plugs were explanted, and alternate sections were stained with hematoxylin-eosin or stained for GFP expression. RESULTS Mean DNA-loaded MB diameter ± standard deviation was 2.87 μm ± 1.69 with the DNA associated with the MB shell. C17.2 cells were associated with 2-4 MBs each, and more than 90% were viable. Peak background subtracted bioluminescent signal was fourfold higher when cells were exposed to 2 W/cm(2) pulses as compared with 1 W/cm(2) pulses (P = .02) and negative controls (P = .002). Histologic examination showed cells within the Matrigel (BD Biosciences) with robust GFP expression only after 2 W/cm(2) ultrasound exposure and lipofectamine transfection. CONCLUSION Stem cells loaded with DNA-carrying MBs can be transfected in vivo with ultrasonic pulses and remain alive to demonstrate robust gene expression.