Brian Walton

Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor

Objectives Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1. Methods and Results Plasma LDL samples isolated from asymptomatic hypercholesterolemic (LDL cholesterol [LDL-C] levels, 154.6±20 mg/dL; n = 7) patients and normocholesterolemic (LDL-C levels, 86.1±21 mg/dL; P<0.001; n = 7) control individuals were chromatographically resolved into 5 subfractions, L1-L5. The L5 percentage (L5%) and the plasma L5 concentration ([L5]  =  L5% × LDL-C) in the patient and control groups were 8.1±2% vs. 2.3±1% (P<0.001) and 12.6±4 mg/dL vs. 1.9±1 mg/dL (P<0.001), respectively. In hypercholesterolemic patients treated with atorvastatin for 6 months (10 mg/day), [L5] decreased from 12.6±4 mg/dL to 4.5±1.1 mg/dL (P = 0.011; n = 5), whereas both [L5] and L5% returned to baseline levels in 2 noncompliant patients 3 months after discontinuation. In cultured human aortic ECs (HAECs), L5 upregulated CRP expression in a dose- and time-dependent manner up to 2.5-fold (P<0.01), whereas the least electronegative subfraction, L1, had no effect. DiI-labeled L1, internalized through the LDL receptor, became visible inside HAECs within 30 seconds. In contrast, DiI-labeled L5, internalized through LOX-1, became apparent after 5 minutes. L5-induced CRP expression manifested at 30 minutes and was attenuated by neutralizing LOX-1. After 30 minutes, L5 but not L1 induced reactive oxygen species (ROS) production. Both L5-induced ROS and CRP production were attenuated by ROS inhibitor N-acetyl cysteine. Conclusions Our results suggest that CRP, L5, and LOX-1 form a cyclic mechanism in atherogenesis and that reducing plasma L5 levels with atorvastatin disrupts the vascular toxicity of L5.

Delivery of negatively charged liposomes into the atherosclerotic plaque of apolipoprotein E-deficient mouse aortic tissue.

Abstract Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular disease. We previously showed the uptake of anionic liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits within lipid pools. However, the cellular distribution of anionic liposomes in atherosclerotic plaque remains undescribed. In addition, how anionic liposomes are absorbed into atherosclerotic plaque is unclear. We investigated the uptake and distribution of anionic liposomes in atherosclerotic plaque in aortic tissues from apolipoprotein E-deficient (ApoE–/–) mice. To facilitate the tracking of liposomes, we used liposomes containing fluorescently labeled non-silencing small interfering RNA. Confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque and colocalization with macrophages. Transmission electron microscopy analysis revealed anionic liposomal accumulation in macrophages. To investigate how anionic liposomes cross the local endothelial barrier, we examined the role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-α. Pretreatment with amantadine, an inhibitor of clathrin-mediated endocytosis, significantly decreased liposomal uptake in HCAECs treated with or without TNF-α by 77% and 46%, respectively. Immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-α but was inhibited by amantadine. These studies indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Our results suggest that anionic liposomes target macrophage-rich areas of vulnerable plaque in ApoE–/– mice; this finding may lead to the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.

Percutaneous intervention for the treatment of hypoplastic aortoiliac syndrome

Hypoplastic aortoiliac syndrome (HAIS) occurs in young women and is characterized by a small infrarenal aorta with a hypoplastic iliofemoral arterial system and advanced atherosclerotic disease. Of 304 aortoiliac interventions (AoI), 30 female patients (mean age, 50 ± 5 years) had HAIS. HAIS patients were less likely to have coronary disease (33% vs. 88%; P < 0.0001) or diabetes (10% vs. 42%; P < 0.001) compared to their AoI counterparts. Twenty‐three patients (73%) had hyperlipidemia with mean cholesterol of 287 ± 42 mg/dl. Twenty‐eight patients (93%) were successfully treated with AoI. The ankle/brachial indices improved from pre‐AoI ABI of 0.55 ± 0.1 to post‐AoI ABI of 0.99 ± 0.1. Complications included one groin hematoma and one case of thrombosis. Follow‐up averaged 31 months (range, 3–91 months) with an early (less than 12 months) restenosis rate of 7% and late (greater than 12 months) restenosis of 21%. One patient (3%) required surgical revascularization for persistent symptoms. Percutaneous treatment of HAIS is an alternative to surgical revascularization with satisfactory long‐term results.Catheter Cardiovasc Interv 2003;60:329–334.

Delivery of negatively charged liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits

Liposomes have been used as imaging and therapeutic agents in various tissues but only infrequently in the cardiovascular system. We prepared a liposome to target atheromas in a Watanabe heritable hyperlipidemic (WHHL) rabbit model. Liposomes labeled with rhodamine and nanogold were injected intra-arterially into the descending thoracic aortas of WHHL rabbits. The arterial segments of interest were perfusion-fixed and evaluated with immunohistochemistry, light microscopy, and electron microscopy. Deconvolution microscopy showed that rhodamine label was concentrated in the plaque shoulder regions of advanced-stage atheromas; however, rhodamine label was not found in adjacent, non-atherosclerotic aorta. Transmission electron microscopy revealed liposome remnants and the highest concentration of nanogold label in lipid-laden areas of atheromas. Liposomes were concentrated in areas of lipoprotein-associated phospholipase A2 expression. We conclude that modified liposomes can be delivered to the shoulder regions of advanced atheromas in WHHL rabbits and may be useful therapeutically for targeting metabolically active plaque.

Simultaneous morphological and biochemical endogenous optical imaging of atherosclerosis

AIMS The aim of this study was to validate novel imaging technology for simultaneous morphological and biochemical endogenous optical imaging of coronary atherosclerotic plaque. METHODS AND RESULTS Optical coherence tomography (OCT) generates high-resolution 3D images of plaque morphology and endogenous fluorescence lifetime imaging microscopy (FLIM) characterizes biochemical composition. Both imaging modalities rely on plaques intrinsic optical characteristics, making contrast agents unnecessary. A multimodal OCT/FLIM system was utilized to generate luminal biochemical maps superimposed on high-resolution (7 µm axial and 13 µm lateral) structural volumetric images. Forty-seven fresh postmortem human coronary segments were imaged: pathological intimal thickening (PIT, n = 26), fibroatheroma (FA, n = 12), thin-cap FA (TCFA, n = 2), and fibrocalcific plaque (CA, n = 7), determined by histopathology. Multimodal images were evaluated, and each plaque identified as PIT, FA, TCFA, or CA based on expert OCT readers, and as having high-lipid (HL), high-collagen (HC), or low-collagen/low-lipid (LCL) luminal composition based on linear discriminant analysis of FLIM. Of 47 plaques, 89.4% (42/47) of the plaques were correctly identified based on OCT/FLIM evaluation using tissue histopathology and immunohistochemistry as the gold standard. Four of the misclassifications corresponded to confusing PIT with HL luminal composition for FA with HL cap. The other corresponded to confusing FA with a HC cap for FA with an LCL cap. CONCLUSION We have demonstrated the feasibility of accurate simultaneous OCT/FLIM morphological and biochemical characterization of coronary plaques at spatial resolutions and acquisition speeds compatible with catheter-based intravascular imaging. The success of this pilot study sets up future development of a multimodal intravascular imaging system that will enable studies that could help improve our understanding of plaque pathogenesis.

Intimacy and a deadly feud: the interplay of autophagy and apoptosis mediated by amino acids

Autophagy (i.e., “self-eating”) and apoptosis (i.e., type I programmed cell death) are essential and intimately involved in molecular, cellular, and whole-body homeostasis in humans and animals. Autophagy has been categorized as a mechanism of intracellular degradation, recycling, defense, and survival. To date, three types of autophagy have been identified: macroautophagy, microautophagy, and chaperone-mediated autophagy. Recent discoveries strongly suggest that macroautophagy also modulates type II programmed cell death under specific circumstances. Autophagy and apoptosis are fundamentally distinct processes, but are interconnected by common stress initiators and intermediate regulators. During the past two decades, the role of amino acid metabolism and signaling in the regulation of apoptosis and autophagy has been intensively studied. In this review, we summarize recent advances in our understanding of the molecular mechanisms that regulate both autophagy and apoptosis in the context of amino acid signaling.

Multimodal optical coherence tomography and fluorescence lifetime imaging with interleaved excitation sources for simultaneous endogenous and exogenous fluorescence

Multimodal imaging probes a variety of tissue properties in a single image acquisition by merging complimentary imaging technologies. Exploiting synergies amongst the data, algorithms can be developed that lead to better tissue characterization than could be accomplished by the constituent imaging modalities taken alone. The combination of optical coherence tomography (OCT) with fluorescence lifetime imaging microscopy (FLIM) provides access to detailed tissue morphology and local biochemistry. The optical system described here merges 1310 nm swept-source OCT with time-domain FLIM having excitation at 355 and 532 nm. The pulses from 355 and 532 nm lasers have been interleaved to enable simultaneous acquisition of endogenous and exogenous fluorescence signals, respectively. The multimodal imaging system was validated using tissue phantoms. Nonspecific tagging with Alexa Flour 532 in a Watanbe rabbit aorta and active tagging of the LOX-1 receptor in human coronary artery, demonstrate the capacity of the system for simultaneous acquisition of OCT, endogenous FLIM, and exogenous FLIM in tissues.

Mining online full-text literature for novel protein interaction discovery

Mining published articles in biology and medicine is a favored means of identifying potential biomarkers in comparison to conventional reviewing process. This is made possible by the development of public literature databases and data mining algorithms. In this article, we present a method to extract novel protein interactions from online full-text articles for biomarker discovery. By evaluating support and confidence metrics, explicit and implicit protein interactions are extracted from corpus of articles. By properly chosen minimum support and confidence, our method maximizes the identification of known interactions while minimizing the number of novel interactions. Hence, our method provides a manageable size of novel interactions for biological validation.

DELIVERY OF LIPOSOMAL FATTY ACID BINDING PROTEIN 4 SIRNA FOR THE TREATMENT OF ATHEROSCLEROSIS IN APOLIPOPROTEIN E-DEFICIENT MICE

Fatty acid binding protein 4 (FABP4) deficiency protects apolipoprotein E-deficient (ApoE−/−) mice against developing atherosclerosis, suggesting that FABP4 may be a potential drug target for treating atherosclerosis. We developed a liposomal siRNA delivery system for silencing FABP4 as a