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Abstract Background Atherosclerosis is an arterial vessel wall disease characterized by slow, progressive lipid accumulation, smooth muscle disorganization, and inflammatory infiltration. Atherosclerosis often remains subclinical until extensive inflammatory injury promotes vulnerability of the atherosclerotic plaque to rupture with luminal thrombosis, which can cause the acute event of myocardial infarction or stroke. Current bioimaging techniques are unable to capture the pathognomonic distribution of cellular elements of the plaque and thus cannot accurately define its structural disorganization. Methods We applied cardiovascular magnetic resonance spectroscopy (CMRS) and diffusion weighted CMR (DWI) with generalized Q-space imaging (GQI) analysis to architecturally define features of atheroma and correlated these to the microscopic distribution of vascular smooth muscle cells (SMC), immune cells, extracellular matrix (ECM) fibers, thrombus, and cholesteryl esters (CE). We compared rabbits with normal chow diet and cholesterol-fed rabbits with endothelial balloon injury, which accelerates atherosclerosis and produces advanced rupture-prone plaques, in a well-validated rabbit model of human atherosclerosis. Results Our methods revealed new structural properties of advanced atherosclerosis incorporating SMC and lipid distributions. GQI with tractography portrayed the locations of these components across the atherosclerotic vessel wall and differentiated multi-level organization of normal, pro-inflammatory cellular phenotypes, or thrombus. Moreover, the locations of CE were differentiated from cellular constituents by their higher restrictive diffusion properties, which permitted chemical confirmation of CE by high field voxel-guided CMRS. Conclusions GQI with tractography is a new method for atherosclerosis imaging that defines a pathological architectural signature for the atheromatous plaque composed of distributed SMC, ECM, inflammatory cells, and thrombus and lipid. This provides a detailed transmural map of normal and inflamed vessel walls in the setting of atherosclerosis that has not been previously achieved using traditional CMR techniques. Although this is an ex-vivo study, detection of micro and mesoscale level vascular destabilization as enabled by GQI with tractography could increase the accuracy of diagnosis and assessment of treatment outcomes in individuals with atherosclerosis.

Abstract Background Brain aging is a major risk factor in the progression of cognitive diseases including Alzheimer’s disease (AD) and vascular dementia. We investigated a mouse model of brain aging up to 24 months old (mo). Methods A high field (11.7T) MRI protocol was developed to characterize specific features of brain aging including the presence of cerebral microbleeds (CMBs), morphology of grey and white matter, and tissue diffusion properties. Mice were selected from age categories of either young (3 mo), middle-aged (18 mo), or old (24 mo) and fed normal chow over the duration of the study. Mice were imaged in vivo with multimodal MRI, including conventional T2-weighted (T2W) and T2*-weighted (T2*W) imaging, followed by ex vivo diffusion-weighted imaging (DWI) and T2*W MR-microscopy to enhance the detection of microstructural features. Results Structural changes observed in the mouse brain with aging included reduced cortical grey matter volume and enlargement of the brain ventricles. A remarkable age-related change in the brains was the development of CMBs found starting at 18 mo and increasing in total volume at 24 mo, primarily in the thalamus. CMBs presence was confirmed with high resolution ex vivo MRI and histology. DWI detected further brain tissue changes in the aged mice including reduced fractional anisotropy, increased radial diffusion, increased mean diffusion, and changes in the white matter fibers visualized by color-coded tractography, including around a large cortical CMB. Conclusions The mouse is a valuable model of age-related vascular contributions to cognitive impairment and dementia (VCID). In composite, these methods and results reveal brain aging in older mice as a multifactorial process including CMBs and tissue diffusion alterations that can be well characterized by high field MRI.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2′,7′-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the “inhibitors” previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Abstract Background and aims Endothelial surface glycocalyx shedding plays a role in endothelial dysfunction and increases vessel wall permeability, which can lead to inflammation and atherogenesis. We sought to elucidate whether a high fat diet (HFD) or disturbed blood flow conditions, both of which are atherogenic risk factors, would contribute more detrimentally to pre-atherosclerotic loss of endothelial glycocalyx integrity and vascular inflammation. Methods Six to seven week-old C57BL/6-background apolipoprotein-E-knockout (ApoE-KO) male mice were either fed a chow diet, fed a modified Western HFD, and/or subjected to a partial left carotid artery (LCA) ligation procedure to induce disturbed blood flow patterns in the LCA. Mice were sacrificed after 1 week of experimental conditions. Both LCA and right carotid artery (RCA) vessels were dissected and preserved to compare glycocalyx coverage and thickness as well as macrophage accumulation in carotid arterial walls amongst and between cohorts. Results Glycocalyx coverage of the endothelium was significantly reduced in the LCAs of HFD fed mice when compared to the control. More significant reduction in glycocalyx coverage occurred in the LCAs of mice exposed to disturbed flow by partial LCA ligation when compared to the control. No differences were found in glycocalyx coverage of RCAs from all cohorts. Regarding inflammation, no difference in macrophage accumulation in carotid arterial walls was observed when comparing the LCAs and RCAs of control and HFD fed mice. However, macrophage infiltration in vessel walls showed a 20-fold increase in the LCAs exposed to disturbed flow following ligation, when compared to control LCAs, while no such statistical difference was observed between the RCAs of the group. Conclusions In our mouse model, endothelial glycocalyx integrity was compromised more by disturbed blood flow patterns than by exposure of the carotid vessel to HFD conditions. The pathophysiological implications include endothelial dysfunction, which correlates to macrophage infiltration in vessel walls and promotes atherogenesis.

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec­topic expression of caveolin-3 also results in enhanced FA sequestration. To further discriminate the effect that caveolins have on transmembrane FA movement and distribution, we labeled the outer membrane leaflet with fluorescein-phosphatidylethanolamine (FPE), whose emission is quenched by the presence of FA anions. Real-time measurements made with FPE and control experiments with positively charged fatty amines support our hypothesis that caveolins promote localization of FA anions through interactions with basic amino acid residues (lysines and arginines) present at the C termini of caveolins-1 and -3.

We performed detailed biophysical studies of transfer of long-chain fatty acids (FAs) from methyl-β-CD (MBCD) to model membranes (egg-PC vesicles) and cells and the extraction of FA from membranes by MBCD. We used i) fluorescein phosphatidylethanolamine to detect transfer of FA anions arriving in the outer membrane leaflet; ii) entrapped pH dyes to measure pH changes after FA diffusion (flip-flop) across the lipid bilayer; and iii) soluble fluorescent-labeled FA binding protein to measure the concentration of unbound FA in water. FA dissociated from MBCD, bound to the membrane, and underwent flip-flop within milliseconds. In the presence of vesicles, MBCD maintained the aqueous concentration of unbound FA at low levels comparable to those measured with albumin. In studies with cells, addition of oleic acid (OA) complexed with MBCD yielded rapid (seconds) dose-dependent OA transport into 3T3-L1 preadipocytes and HepG2 cells. MBCD extracted OA from cells and model membranes rapidly at concentrations exceeding those required for OA delivery but much lower than concentrations commonly used for extracting cholesterol. Compared with albumin, MBCD can transfer its entire FA load and is less likely to extract cell nutrients and to introduce impurities.

Disruption and thrombosis of atherosclerotic plaques cause most acute cardiovascular events, but their systematic study has been hampered by the lack of suitable animal models. To assess the value of a modified rabbit model of atherothrombosis, we performed detailed histology of rabbit aortic plaques. Atherosclerosis was induced with a high cholesterol diet fed 2 weeks prior to and 6 weeks after balloon injury of the aorta, followed by 4 weeks of normal diet. We found six out of eight types of plaques cataloged by the American Heart Association in the rabbit aorta. Vulnerable plaques were defined as those with attached platelet and fibrin-rich thrombi after pharmacological triggering with Russell's viper venom and histamine. Ruptured plaques had, as also described for human plaques: i) marked medial and adventitial changes, including neovascularization and inflammation; ii) cholesterol monohydrate crystals and liquid crystalline cholesterol esters in the intima and the fibrous cap; and iii) inflamed, thin fibrous caps. Increased cholesterol monohydrate area, internal elastic lamina area, positive remodeling, fibrous cap inflammation, adventitia breakdown, and inflammation were independent predictors of plaque disruption. Our findings reveal novel insights into plaque vulnerability and could guide the design of noninvasive imaging approaches for detecting and treating high-risk plaques.

α-Synuclein (αS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. Although unstructured in solution, αS forms α-helices in the presence of negatively charged lipid surfaces. Moreover, αS was shown to interact with FAs in a manner that promotes protein aggregation. Here, we investigate whether αS has specific FA binding site(s) similar to fatty acid binding proteins (FABPs), such as the intracellular FABPs. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of αS signal in both 1H and 13C spectra and ii) the appearance of a very broad FA 13C-carboxyl signal. These data exclude high-affinity binding of FA molecules to specific αS sites, as in FABPs. One possible mode of binding was revealed by electron microscopy studies of oleic acid bilayers at pH 7.8; these high-molecular-weight FA aggregates possess a net negative surface charge because they contain FA anions, and they were easily disrupted to form smaller particles in the presence of αS, indicating a direct protein-lipid interaction. We conclude that αS is not likely to act as an intracellular FA carrier. Binding to negatively charged membranes, however, appears to be an intrinsic property of αS that is most likely related to its physiological role(s) in the cell.

Vulnerable atherosclerotic plaques may be identified by their large lipid component, particularly liquid cholesteryl ester (CE), covered by a fibrous cap. We hypothesized that image-guided 1H proton magnetic resonance spectroscopy (MRS) would identify mobile CE in discrete, preselected regions of atherosclerotic plaque. Human carotid endarterectomy specimens (n = 10) were imaged ex vivo by magnetic resonance imaging (MRI) at high field (11.7 T) utilizing standard T1- and T2-weighted spin echo protocols. MRS spectra were acquired from 1 mm3 voxels, localized to plaque regions that we judged by MRI to be lipid rich or lipid poor. The spectra revealed methyl and methylene resonances of fatty acyl chains with relative intensities and linewidths characteristic of pure CE, by comparison with lipid standards. Regions judged to be lipid rich by MRI showed much more intense CE resonances than did lipid-poor regions. The integrated intensities of lipid peaks were 5.5 ± 2.0% (lipid-rich regions) versus 0.9 ± 0.6% (lipid-poor regions) of the unsuppressed water peak (P < 0.0001). Lipid distribution by histology, MRS, and MRI showed strong correlation. Image-guided proton MRS accurately identified CE in selected regions of atherosclerotic plaque as small as 1 mm3 in an ex vivo setting. This procedure may permit the noninvasive detection and quantification of CE in atherosclerotic plaque in vivo.

Esterification of fatty acids with the small polar molecule carnitine is a required step for the regulated flow of fatty acids into mitochondrial inner matrix. We have studied the interactions of acyl carnitines (ACs) with model membranes [egg yolk phosphatidylcholine (PC) vesicles] by 13C-nuclear magnetic resonance (NMR) spectroscopy. Using AC with 13C-enrichment of the carbonyl carbon of the acyl chain, we detected NMR signals from AC on the inside and outside leaflets of the bilayer of small unilamellar vesicles prepared by cosonication of PC and AC. However, when AC was added to the outside of pre-formed PC vesicles, only the signal for AC bound to the outer leaflet was observed, even after hours at equilibrium. The extremely slow transmembrane diffusion (“flip-flop”) is consistent with the zwitterionic nature of the carnitine head group and the known requirement of transport proteins for movement of ACs through the mitochondrial membrane. The partitioning of ACs (8–18 carbons) between water and PC vesicles was studied by monitoring the [13C]carbonyl chemical shift of ACs as a function of pH and concentration of vesicles. Significant partitioning into the water phase was detected for ACs with chain lengths of 12 carbons or less. The effect of ACs on the integrity of the bilayer was examined in vesicles with up to 25 mol% myristoyl carnitine; no gross disruption of the bilayer was observed.We hypothesize that the effects of high levels of long-chain AC (as found in ischemia or in certain diseases) on cell membranes result from molecular effects on membrane functions rather than from gross disruption of the lipid bilayer.

The remarkable binding properties of serum albumin have been investigated extensively, but little is known about an important class of fatty acids, the very long-chain saturated fatty acids (VLCFA; >18 carbons). Although VLCFA are metabolized efficiently in normal individuals, they are markers for and possibly causative agents of several peroxisomal disorders. We studied the binding of [13C]carboxyl-enriched arachidic (C20:0), behenic (C22:0), lignoceric (C24:0), and hexacosanoic (C26:0) acids to bovine serum albumin (BSA) by 13C-NMR spectroscopy. For each VLCFA, the NMR spectra showed multiple signals at chemical shifts previously identified for long-chain fatty acids (12–18 carbons), suggesting stabilization of binding by similar, if not identical, interactions of the fatty acid carboxyl anion with basic amino acid residues. The maximal binding (mol of VLCFA/mol of BSA) and the number of observed binding sites decreased with increasing chain length, from 4–5 for C20:0, 3–4 for C22:0, and 2 for C24:0; we validated our previous conclusion that BSA has only one site for C26:0 (Ho, J. K., H. Moser, Y. Kishimoto, and J. A. Hamilton. 1995. J. Clin. Invest. 96: 1455–1463). Analysis of chemical shifts suggested that the highest affinity sites for VLCFA are low affinity sites for long-chain fatty acids. In competition experiments with 13C-labeled C22:0 (3 mol/mol of BSA) and unlabeled oleic acid, C22:0 bound to BSA in the presence of up to 4 mol of oleic acid/mol of BSA, but 1 mol was shifted into a different site.Our studies suggest that albumin has adequate binding capacity for the low plasma levels of VLCFA with 20 to 26 carbons, but the protein may not be able to bind longer chain VLCFA.

Transport of unesterified fatty acids (FA) into cells has been viewed either as a simple diffusion process regulated mainly by lipid physical chemistry or as a more complex process involving protein catalysis. In this review FA transport in cell membranes is broken down into three essential steps: adsorption, transmembrane movement, and desorption. The physical properties of FA in aqueous, membrane, and protein environments relevant to transport mechanisms are discussed, with emphasis on recent information derived from NMR and fluorescence studies. Because of their low solubility in water and high hydrophobicity, FA bind rapidly and avidly to model membranes (phospholipid bilayers); if albumin is a donor, FA desorb rapidly to reach their equilibrium distribution between the membrane and albumin. The ionization properties of FA in a phospholipid bilayer result in a high population of the un-ionized form (∼50%) at pH 7.4, which diffuses across the lipid bilayer (flip-flops) rapidly (t1/2 < 1 sec). Desorption of FA from a phospholipid surface is slower than transmembrane movement and dependent on the FA chain length and unsaturation, but is rapid for typical dietary FA. These physical properties of FA in model systems predict that proteins are not essential for transport of FA through membranes. The only putative FA transport protein to be purified and reconstituted into phospholipid bilayers, the mitochondrial uncoupling protein (UCP1), was shown to transport the FA anion in response to FA flip-flop. New experiments with cells have found that FA movement into cells acidifies the cytosol, as predicted by the flip-flop model.—Hamilton, J. A. Fatty acid transport: difficult or easy?

Edible crabs (Cancer pagurus) are an economically important species for Scottish inshore fisheries, with an estimated annual landing value of GBP 16 million (2023). Research into the behaviour, particularly agonistic behaviour, of this species is currently lacking. This paper aims to investigate behaviour, social interactions, potential hierarchies, and the impact of claw size on the outcomes of agonistic interactions of male C. pagurus through behavioural trials and retrials. Crabs were semi-randomly assigned to pairs (based on allocated condition index rating) and introduced to one another in trial tanks. Each pair underwent two trials, 24 h apart (the “trial” and “retrial”). Analyses of video records of agonistic bouts revealed that 77% of retrials were won by the initial victors, with a significant decrease in fight time between trials and retrials. Fight time was not correlated with weapon size (claw length, height, and depth). There were no differences in weapon size of winners and losers of bouts. Winners exhibited a significantly higher frequency of aggressive and dominant behaviours (approach, aggressive contact, threat displays, and mounting), and losers exhibited higher frequencies of submissive behaviours (withdrawal, retracting limbs, and remaining motionless). These results suggest that individual behaviour may play more of a role in dominance than size or other morphometric characteristics.

Summary: Non-alcoholic steatohepatitis (NASH) is a most common chronic liver disease that is manifested by steatosis, inflammation, fibrosis, and tissue damage. Hepatocytes produce giant mitochondria termed megamitochondria in patients with NASH. It has been shown that gene knockout of OPA1, a mitochondrial dynamin-related GTPase that mediates mitochondrial fusion, prevents megamitochondria formation and liver damage in a NASH mouse model induced by a methionine-choline-deficient (MCD) diet. However, it is unknown whether blocking mitochondrial fusion mitigates NASH pathologies. Here, we acutely depleted OPA1 using antisense oligonucleotides in the NASH mouse model before or after megamitochondria formation. When OPA1 ASOs were applied at the disease onset, they effectively prevented megamitochondria formation and liver pathologies in the MCD model. Notably, even when applied after mice robustly developed NASH pathologies, OPA1 targeting effectively regressed megamitochondria and the disease phenotypes. Thus, our data show the efficacy of mitochondrial dynamics as a unique therapy for megamitochondria-associated liver disease.

The use of unmanned aerial vehicles (UAVs—commonly referred to as drones) in journalism has emerged only recently, and has grown significantly. This article explores what makes drone imagery as an instance of what scholars of visual culture call an aerial view so compelling for major news organizations as to warrant such attention and investment. To do this, the concept ‘visual aggregation’ is introduced to theorize the authority of drone imagery in conventional journalistic practice. Imagery produced through drone journalism is a visual analogy to statistical summary and, more recently, of what is referred to as data journalism. Just as these combine an aggregate of cases to produce an understanding of an overall trend, drone imagery aggregates space visually, its broad visual field revealing large-scale spatial patterns in ways analogous to the statistical capture/analysis of large bodies of data. The article then employs a cultural and historical approach to identify key points in the emergence of visual aggregation as authoritative truth. The aerial view as a claim to truth is manifest in a wide range of antecedent social formations, devices and practices prior to their amalgamation in what has today become drone journalism. This analysis aids understanding of how drone journalism is a response to the institutional crises of journalism today.