Research Priorities in Percutaneous Image- and Endoscopy-Guided Interventions for Biliary and Gallbladder Diseases: Proceedings from the Society of Interventional Radiology Foundation Multidisciplinary Research Consensus Panel.

Recent technological advancements, including the introduction of disposable endoscopes, have enhanced the role of interventional radiology (IR) in the management of biliary and gallbladder diseases. There are unanswered questions in this growing field. The Society of Interventional Radiology Foundation convened a virtual research consensus panel consisting of a multidisciplinary group of experts to develop a prioritized research agenda regarding percutaneous image- and endoscopy-guided procedures for biliary and gallbladder diseases. The panelists discussed current data, opportunities for IR, and future efforts to maximize IR's ability and scope. A recurring theme throughout the discussions was to find ways to reduce the total duration of percutaneous drains and improve patients' quality of life. After the presentations and discussions, research priorities were ranked on the basis of their clinical relevance and impact. The research ideas ranked top 3 were as follows: (a) percutaneous multimodality management of benign anastomotic biliary strictures (laser vs endobiliary ablation vs cholangioplasty vs drain upsize protocol alone), (b) ablation of intraductal cholangiocarcinoma with and without stent placement, and (c) cholecystoscopy/choledochoscopy and lithotripsy in nonsurgical patients with calculous cholecystitis. Collaborative, retrospective, and prospective research studies are essential to answer these questions and improve the management protocols for patients with biliary and gallbladder diseases.

The Role of Physician-Driven Device Preference in the Cost Variation of Common Interventional Radiology Procedures.

PURPOSE: To analyze the impact of physician-specific equipment preference on cost variation for procedures typically performed by interventional radiologists at a tertiary care academic hospital. MATERIALS AND METHODS: From October 2017 to October 2019, data on all expendable items used by 9 interventional radiologists for 11 common interventional radiology procedure categories were compiled from the hospital analytics system. This search yielded a final dataset of 44,654 items used in 2,121 procedures of 11 different categories. The mean cost per case for each physician as well as the mean, standard deviation, and coefficient of variation (CV) of the mean cost per case across physicians were calculated. The proportion of spending by item type was compared across physicians for 2 high-variation, high-volume procedures. The relationship between the mean cost per case and case volume was examined using linear regression. RESULTS: There was a high variability within each procedure, with the highest and the lowest CV for radioembolization administration (56.6%) and transjugular liver biopsy (4.9%), respectively. Variation in transarterial chemoembolization cost was mainly driven by microcatheters/microwires, while for nephrostomy, the main drivers were catheters/wires and access sets. Mean spending by physician was not significantly correlated with case volume (P =.584). CONCLUSIONS: Physicians vary in their item selection even for standard procedures. While the financial impact of these differences vary across procedures, these findings suggest that standardization may offer an opportunity for cost savings.

Comparison of Opioid Medication Use after Conventional Chemoembolization versus Drug-Eluting Embolic Chemoembolization.

PURPOSE: To assess the use of opioid analgesics and/or antiemetic drugs for pain and nausea following selective chemoembolization with doxorubicin-based conventional (c)-transarterial chemoembolization versus drug-eluting embolic (DEE)-transarterial chemoembolization for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: From October 2014 to 2016, 283 patients underwent 393 selective chemoembolization procedures including 188 patients (48%) who underwent c-transarterial chemoembolization and 205 (52%) who underwent DEE-transarterial chemoembolization. Medical records for all patients were retrospectively reviewed. Administration of postprocedural opioid and/or antiemetic agents were collated. Time of administration was stratified as phase 1 recovery (0-6 hours) and observation (6-24 hours). Logistic regression model was used to investigate the relationship of transarterial chemoembolization type and use of intravenous and/or oral analgesic and antiemetic medications while controlling for other clinical variables. RESULTS: More patients treated with DEE-transarterial chemoembolization required intravenous analgesia in the observation (6-24 hours) phase (18.5%) than those treated with c-transarterial chemoembolization (10.6%; P = .033). Similar results were noted for oral analgesic agents (50.2% vs. 31.4%, respectively; P < .001) and antiemetics (17.1% vs. 7.5%, respectively; P = .006) during the observation period. Multivariate regression models identified DEE-transarterial chemoembolization as an independent predictor for oral analgesia (odds ratio [OR], 1.84; P = .011), for intravenous and oral analgesia in opioid-naïve patients (OR, 2.46; P = .029) and for antiemetics (OR, 2.56; P = .011). CONCLUSIONS: Compared to c-transarterial chemoembolization, DEE-transarterial chemoembolization required greater amounts of opioid analgesic and antiemetic agents 6-24 hours after the procedure. Surgical data indicate that a persistent opioid habit can develop even after minor surgeries, therefore, caution should be exercised, and a regimen of nonopiate pain medications should be considered to reduce postprocedural pain after transarterial chemoembolization.

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.

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.

apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL.

The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.

Single-shot quantitative x-ray imaging using a primary modulator and dual-layer detector.

BACKGROUND: Conventional x-ray imaging and fluoroscopy have limitations in quantitation due to several challenges, including scatter, beam hardening, and overlapping tissues. Dual-energy (DE) imaging, with its capability to quantify area density of specific materials, is well-suited to address such limitations, but only if the dual-energy projections are acquired with perfect spatial and temporal alignment and corrected for scatter. PURPOSE: In this work, we propose single-shot quantitative imaging (SSQI) by combining the use of a primary modulator (PM) and dual-layer (DL) detector, which enables motion-free DE imaging with scatter correction in a single exposure. METHODS: The key components of our SSQI setup include a PM and DL detector, where the former enables scatter correction for the latter while the latter enables beam hardening correction for the former. The SSQI algorithm allows simultaneous recovery of two material-specific images and two scatter images using four sub-measurements from the PM encoding. The concept was first demonstrated using simulation of chest x-ray imaging for a COVID patient. For validation, we set up SSQI geometry on our tabletop system and imaged acrylic and copper slabs with known thicknesses (acrylic: 0-22.5 cm; copper: 0-0.9 mm), estimated scatter with our SSQI algorithm, and compared the material decomposition (MD) for different combinations of the two materials with ground truth. Second, we imaged an anthropomorphic chest phantom containing contrast in the coronary arteries and compared the MD with and without SSQI. Lastly, to evaluate SSQI in dynamic applications, we constructed a flow phantom that enabled dynamic imaging of iodine contrast. RESULTS: Our simulation study demonstrated that SSQI led to accurate scatter correction and MD, particularly for smaller focal blur and finer PM pitch. In the validation study, we found that the root mean squared error (RMSE) of SSQI estimation was 0.13 cm for acrylic and 0.04 mm for copper. For the anthropomorphic phantom, direct MD resulted in incorrect interpretation of contrast and soft tissue, while SSQI successfully distinguished them quantitatively, reducing RMSE in material-specific images by 38%-92%. For the flow phantom, SSQI was able to perform accurate dynamic quantitative imaging, separating contrast from the background. CONCLUSIONS: We demonstrated the potential of SSQI for robust quantitative x-ray imaging. The integration of SSQI is straightforward with the addition of a PM and upgrade to a DL detector, which may enable its widespread adoption, including in techniques such as radiography and dynamic imaging (i.e., real-time image guidance and cone-beam CT).

Design and implementation of a cost-effective, open-source, and programmable pulsatile flow system.

The primary objective of this research was to design, implement, and validate a programmable open-source pulsatile flow system to cost-effectively simulate vascular flows. We employed an Arduino-compatible microcontroller combined with a motor driver to control a centrifugal direct current (DC) motor pump. The system was programmed to produce pulsatile flows with an arterial pulse waveform. Validation with Doppler ultrasound and flow measurements confirmed that our Arduino-based system successfully replicated arterial vascular flow. The materials are easily accessible, with a total bill of materials as low as $99. This open-source programmable pulsatile pump platform offers superior cost-effectiveness and adaptability relative to commercial offerings.

Label-free monitoring of embolotherapy via catheter electrochemical impedance spectroscopy.

Catheter-based embolization has become a widely adopted minimally-invasive treatment for a broad range of applications. However, assessment of embolization endpoints requires x-ray fluoroscopic monitoring, exposing patients and physicians performing embolization procedures to harmful ionizing radiation. Moreover, x-ray fluoroscopy assessment of embolization endpoints is low sensitivity, subjective, and may not reflect the actual physiology of blood flow reduction, thus providing little oversight of the embolization procedure. Inspired by the observation that the dielectric properties of blood differ from those of fluids injected during the embolization procedure, a customized angiographic catheter was created with embedded electrodes for catheter-based electrochemical impedance spectroscopy as a way to monitor embolization. Real-time electrochemical impedance spectroscopy was performed in a phantom and compared to visual and videographic monitoring. Electrochemical impedance spectroscopy was able to sense endpoints of embolization, including stasis, reflux, and persistent flow. This new technique offers a label-free method of sensing embolization progress with potentially higher sensitivity and reproducibility compared to x-ray, as well as offer substantial reduction in x-ray exposure to patients and physicians.

A2b adenosine receptor regulates hyperlipidemia and atherosclerosis.

BACKGROUND: The cAMP-elevating A(2b) adenosine receptor (A(2b)AR) 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 A(2b)AR. Bone marrow transplantation experiments indicated that A(2b)AR bone marrow cell signals alone were not sufficient to elicit this effect. Intriguingly, liver expression of the A(2b)AR in wild-type mice was vastly augmented by a high-fat diet, raising the possibility that this upregulation is of functional significance. A(2b)AR 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 A(2b)AR-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 A(2b)AR in primary hepatocytes confirmed the regulation of SREBP-1 by this receptor. A(2b)AR-mediated changes in cAMP were found to regulate levels of the transcriptionally active form of SREBP-1. Finally, adenovirally mediated restoration of the A(2b)AR in the liver of A(2b)AR-null mice reduced the lipid profile and atherosclerosis. Similarly, in vivo administration of the A(2b)AR 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 A(2b)AR regulates liver SREBP-1, hyperlipidemia, and atherosclerosis, suggesting that this receptor may be an effective therapeutic target.

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.

Percutaneous Cryoneurolysis for Pain Control After Rib Fractures in Older Adults.

This case series evaluates changes in numeric pain scores, opioid use, and other measures before, during, and 30 days after computed tomography­guided percutaneous cryoneurolysis in patients with rib injury.

Fluorous-phase iron oxide nanoparticles as enhancers of acoustic droplet vaporization of perfluorocarbons with supra-physiologic boiling point.

Perfluorocarbon emulsion nanodroplets containing iron oxide nanoparticles (IONPs) within their inner perfluorohexane (PFH) core were prepared to investigate potential use as an acoustically activatable ultrasound contrast agent, with the hypothesis that incorporation of IONPs into the fluorous phase of a liquid perfluorocarbon emulsion would potentiate acoustic vaporization. IONPs with an oleic acid (OA) hydrophobic coating were synthesized through chemical co-precipitation. To suspend IONP in PFH, OA was exchanged with perfluorononanoic acid (PFNA) via ligand exchange to yield fluorophilic PFNA-coated IONPs (PFNA-IONPs). Suspensions with various amounts of PFNA-IONPs (0-15% w/v) in PFH were emulsified in saline by sonication, using 5% (w/v) egg yolk phospholipid as an emulsifier. PFNA-IONPs were characterized with transmission electron microscopy (TEM), transmission electron cryomicroscopy (cryoTEM), and thermogravimetric analysis (TGA) with Fourier transform infrared spectroscopy (FTIR). IONP were between 5 and 10 nm in diameter as measured by electron microscopy, and hydrodynamic size of the PFH nanodroplets were 150 to 230 nm as measured by dynamic light scattering (DLS). Acoustic droplet vaporization of PFH nanodroplets (PFH-NDs) was induced using conversion pulses (100 cycle at 1.1 MHz and 50% duty cycle) provided by a focused ultrasound transducer, and formed microbubbles were imaged using a clinical ultrasound scanner. The acoustic pressure threshold needed for PFH-NDs vaporization decreased with increasing temperature and IONP content. PFH-NDs containing 5% w/v IONP converted to microbubbles at 42 °C at 2.18 MI, which is just above the exposure limits of 1.9 MI allowed by the FDA for clinical ultrasound scanners, whereas 10 and 15% emulsion vaporized at 1.87 and 1.24 MI, respectively. Furthermore, 5% IONP-loaded PFH-NDs injected intravenously into melanoma-bearing mice at a dose of 120 mg PFH/kg, converted into detectable microbubbles in vivo 5 h, but not shortly after injection, indicating that this technique detects NDs accumulated in tumors.

Contrast-enhanced ultrasound of the liver: technical and lexicon recommendations from the ACR CEUS LI-RADS working group.

Contrast-enhanced ultrasound (CEUS) is a specific form of ultrasound imaging performed with intravenous administration of microbubble contrast agents. It has been extensively used for liver tumor characterization and was recently added to the American College of Radiology Liver Imaging Reporting and Data System (CEUS LI-RADS). This paper describes technical recommendations for successful liver CEUS lesion characterization, and provides imaging protocol and Lexicon of imaging findings.

Thrombin-Activatable Microbubbles as Potential Ultrasound Contrast Agents for the Detection of Acute Thrombosis.

Acute deep vein thrombosis (DVT) is the formation of a blood clot in the deep veins of the body that can lead to fatal pulmonary embolism. Acute DVT is difficult to distinguish from chronic DVT by ultrasound (US), the imaging modality of choice, and is therefore treated aggressively with anticoagulants, which can lead to internal bleeding. Here we demonstrate that conjugating perfluorobutane-filled (PFB-filled) microbubbles (MBs) with thrombin-sensitive activatable cell-penetrating peptides (ACPPs) could lead to the development of contrast agents that detect acute thrombosis with US imaging. Successful conjugation of ACPP to PFB-filled MBs was confirmed by fluorescence microscopy and flow cytometry. Fluorescein-labeled ACPP was used to evaluate the efficiency of thrombin-triggered cleavage by measuring the mean fluorescence intensity of ACPP-labeled MBs (ACPP-MBs) before and after incubation at 37 °C with thrombin. Lastly, control MBs and ACPP-MBs were infused through a tube containing a clot, and US contrast enhancement was measured with or without the presence of a thrombin inhibitor after washing the clot with saline. With thrombin activity, 91.7 ± 14.2% of the signal was retained after ACPP-MB infusion and washing, whereas only 16.7 ± 4% of the signal was retained when infusing ACPP-MBs in the presence of hirudin, a potent thrombin inhibitor.

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.

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.

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

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.

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 molecule's 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.