Lipid and smooth muscle architectural pathology in the rabbit atherosclerotic vessel wall using Q-space cardiovascular magnetic resonance.

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.

SSO and other putative inhibitors of FA transport across membranes by CD36 disrupt intracellular metabolism, but do not affect FA translocation.

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.

The brains of aged mice are characterized by altered tissue diffusion properties and cerebral microbleeds.

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.

Concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model.

The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood-brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or electrophysiological dysfunction. Furthermore, concussion-like deficits were observed after impact injury, but not after blast exposure under experimental conditions matched for head kinematics. Computational modelling showed that impact injury generated focal point loading on the head and seven-fold greater peak shear stress in the brain compared to blast exposure. Moreover, intracerebral shear stress peaked before onset of gross head motion. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that force loading mechanics at the time of injury shape acute neurobehavioural responses, structural brain damage, and neuropathological sequelae triggered by neurotrauma. These results indicate that closed-head impact injuries, independent of concussive signs, can induce traumatic brain injury as well as early pathologies and functional sequelae associated with chronic traumatic encephalopathy. These results also shed light on the origins of concussion and relationship to traumatic brain injury and its aftermath.awx350media15713427811001.

Atherosclerosis, Periodontal Disease, and Treatment with Resolvins.

PURPOSE OF REVIEW: This review aims to discuss the existing evidence on the link between atherosclerosis and periodontitis by particularly presenting new findings that link the pathology and therapy of these diseases. Acute vascular ischemic events that can lead to stroke or myocardial infarction are initiated by inflammatory processes leading to rupture or erosion of plaques susceptible to thrombosis ("high risk" or "vulnerable"). These are highly inflamed plaques residing in the media and adventitia that may not be detected by angiography measurments of luminal narrowing. Statistically significant excess risk for atherosclerotic cardiovascular disease has been reported in persons with periodontitis independent of established risk factors. We hypothesized that the systemic pathologic links also represent potential therapeutic links. RECENT FINDINGS: We recently demonstrated that periodontal inflammation promotes atherosclerotic plaque inflammation and destabilization. As discrete pathological regions, these plaques with a high susceptibility to rupture can be imaged and differentiated from lower risk plaques. In cholesterol-fed rabbits with periodontal disease, circulating inflammatory mediators were also significantly elevated thereby contributing to "vulnerable blood," a systemic characteristic of high risk for cardiovascular events. New studies show that certain lipid mediators, including lipoxins and resolvins, are potent in preventing and possibly treating a number of inflammation-associated diseases, including periodontitis and vascular inflammation. The concept of the vulnerable patient and the pro-resolving approach open new terrain for discovery of paradigm-changing therapies for the prevention and treatment of two of the most common diseases of man. Importantly, lipoxins and resolvins are natural receptor agonists that do not exhibit the same pro-atherogenic side effects attributed to anti-inflammatory medications (e.g., NSAIDs) but rather coordinate resolution of inflammation and a return to homeostasis.

Disorder Amidst Membrane Order: Standardizing Laurdan Generalized Polarization and Membrane Fluidity Terms.

Membrane organization and fluidity research continues to expand and the understanding of membrane dynamics continues to be refined. Within this field of study, laurdan remains among the most popular, versatile, and established fluorescence probes. Fluorimetry and multiphoton microscopy techniques are standards for measuring laurdan fluorescence and continue to be refined. However, complications have arisen due to an amended membrane model, revised terms used for describing membrane phases, and wide variation in the selection of laurdan generalized polarization equation values. Here, in the context of the history and chemical properties of laurdan, discrepancies are highlighted and important recommendations are made to promote uniformity and ongoing progress.

CD36 binds oxidized low density lipoprotein (LDL) in a mechanism dependent upon fatty acid binding.

The association of unesterified fatty acid (FA) with the scavenger receptor CD36 has been actively researched, with focuses on FA and oxidized low density lipoprotein (oxLDL) uptake. CD36 has been shown to bind FA, but this interaction has been poorly characterized to date. To gain new insights into the physiological relevance of binding of FA to CD36, we characterized FA binding to the ectodomain of CD36 by the biophysical method surface plasmon resonance. Five structurally distinct FAs (saturated, monounsaturated (cis and trans), polyunsaturated, and oxidized) were pulsed across surface plasmon resonance channels, generating association and dissociation binding curves. Except for the oxidized FA HODE, all FAs bound to CD36, with rapid association and dissociation kinetics similar to HSA. Next, to elucidate the role that each FA might play in CD36-mediated oxLDL uptake, we used a fluorescent oxLDL (Dii-oxLDL) live cell assay with confocal microscopy imaging. CD36-mediated uptake in serum-free medium was very low but greatly increased when serum was present. The addition of exogenous FA in serum-free medium increased oxLDL binding and uptake to levels found with serum and affected CD36 plasma membrane distribution. Binding/uptake of oxLDL was dependent upon the FA dose, except for docosahexaenoic acid, which exhibited binding to CD36 but did not activate the uptake of oxLDL. HODE also did not affect oxLDL uptake. High affinity FA binding to CD36 and the effects of each FA on oxLDL uptake have important implications for protein conformation, binding of other ligands, functional properties of CD36, and high plasma FA levels in obesity and type 2 diabetes.

Distinct lipid a moieties contribute to pathogen-induced site-specific vascular inflammation.

Several successful pathogens have evolved mechanisms to evade host defense, resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory bone loss and systemic inflammation manifested as atherosclerosis. P. gingivalis expresses an atypical lipopolysaccharide (LPS) structure containing heterogeneous lipid A species, that exhibit Toll-like receptor-4 (TLR4) agonist or antagonist activity, or are non-activating at TLR4. In this study, we utilized a series of P. gingivalis lipid A mutants to demonstrate that antagonistic lipid A structures enable the pathogen to evade TLR4-mediated bactericidal activity in macrophages resulting in systemic inflammation. Production of antagonistic lipid A was associated with the induction of low levels of TLR4-dependent proinflammatory mediators, failed activation of the inflammasome and increased bacterial survival in macrophages. Oral infection of ApoE(-/-) mice with the P. gingivalis strain expressing antagonistic lipid A resulted in vascular inflammation, macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain producing exclusively agonistic lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11-dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE(-/-) mice infected with this strain exhibited diminished vascular inflammation, macrophage accumulation, and atherosclerosis progression. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. Collectively, our results point to a pivotal role for activation of the non-canonical inflammasome in P. gingivalis infection and demonstrate that P. gingivalis evades immune detection at TLR4 facilitating chronic inflammation in the vasculature. These studies support the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.

NMR reveals molecular interactions and dynamics of fatty acid binding to albumin.

BACKGROUND: The molecular details of fatty acid (FA) interactions with albumin are fundamental to understanding transport in the plasma and cellular utilization of these key nutrients and building blocks of membranes. SCOPE OF REVIEW: This review focuses on the development and application of NMR methods to study FA binding to albumin [bovine (BSA) and human (HSA)]. The key strategy was to use (13)C enrichment of a specific carbon in the FA as a non-perturbing probe to permit visualization of the small ligand complexed to the very large protein. NMR contributions to illuminating molecular interactions and FA dynamics are summarized from three decades of studies. MAJOR CONCLUSIONS: Our early studies detected multiple binding sites that we hypothesized were distinguished because of the unique tertiary structure of the protein in close proximity to the FA labeled carbon in each site. Later crystallographic structures revealed the presence of polar and charged amino acid side chains near the carboxyl carbon of the FA and unique tertiary structures lining all of the FA binding pockets. In collaboration with the crystallography group, several FA sites in the crystalline state were matched with NMR resonances in the solution state. With the newest application of NMR, 2D NMR spectroscopy detected nine binding sites, and three were located in the crystal structure through displacement of drugs with identified sites. GENERAL SIGNIFICANCE: NMR spectroscopy utilizing the FA as a probe allows characterization of site-specific interactions, molecular motions within binding sites, the order of filling and removal of FA from sites. This article is part of a Special Issue entitled Serum Albumin.

The influence of pericardial fat upon left ventricular function in obese females: evidence of a site-specific effect.

BACKGROUND: Although increased volume of pericardial fat has been associated with decreased cardiac function, it is unclear whether this association is mediated by systemic overall obesity or direct regional fat interactions. We hypothesized that if local effects dominate, left ventricular (LV) function would be most strongly associated with pericardial fat that surrounds the left rather than the right ventricle (RV). METHODS: Female obese subjects (n = 60) had cardiovascular magnetic resonance (CMR) scans to obtain measures of LV function and pericardial fat volumes. LV function was obtained using the cine steady state free precession imaging in short axis orientation. The amount of pericardial fat was determined volumetrically by the cardiac gated T1 black blood imaging and normalized to body surface area. RESULTS: In this study cohort, LV fat correlated with several LV hemodynamic measurements including cardiac output (r = -0.41, p = 0.001) and stroke volume (r = -0.26, p = 0.05), as well as diastolic functional parameters including peak-early-filling rate (r = -0.38, p = 0.01), early late filling ratio (r = -0.34, p = 0.03), and time to peak-early-filling (r = 0.34, p = 0.03). These correlations remained significant even after adjusting for the body mass index and the blood pressure. However, similar correlations became weakened or even disappeared between RV fat and LV function. LV function was not correlated with systemic plasma factors, such as C-reactive protein (CRP), B-type natriuretic peptide (BNP), Interleukin-6 (IL-6), resistin and adiponectin (all p > 0.05). CONCLUSIONS: LV hemodynamic and diastolic function was associated more with LV fat as compared to RV or total pericardial fat, but not with systemic inflammatory markers or adipokines. The correlations between LV function and pericardial fat remained significant even after adjusting for systemic factors. These findings suggest a site-specific influence of pericardial fat on LV function, which could imply local secretion of molecules into the underlying tissue or an anatomic effect, both mechanisms meriting future evaluation.

Protective role for TLR4 signaling in atherosclerosis progression as revealed by infection with a common oral pathogen.

Clinical and epidemiological studies have implicated chronic infections in the development of atherosclerosis. It has been proposed that common mechanisms of signaling via TLRs link stimulation by multiple pathogens to atherosclerosis. However, how pathogen-specific stimulation of TLR4 contributes to atherosclerosis progression remains poorly understood. In this study, atherosclerosis-prone apolipoprotein-E null (ApoE(-/-)) and TLR4-deficient (ApoE(-/-)TLR4(-/-)) mice were orally infected with the periodontal pathogen Porphyromonas gingivalis. ApoE(-/-)TLR4(-/-) mice were markedly more susceptible to atherosclerosis after oral infection with P. gingivalis. Using live animal imaging, we demonstrate that enhanced lesion progression occurs progressively and was increasingly evident with advancing age. Immunohistochemical analysis of lesions from ApoE(-/-)TLR4(-/-) mice revealed an increased inflammatory cell infiltrate composed primarily of macrophages and IL-17 effector T cells (Th17), a subset linked with chronic inflammation. Furthermore, enhanced atherosclerosis in TLR4-deficient mice was associated with impaired development of Th1 immunity and regulatory T cell infiltration. In vitro studies suggest that the mechanism of TLR4-mediated protective immunity may be orchestrated by dendritic cell IL-12 and IL-10, which are prototypic Th1 and regulatory T cell polarizing cytokines. We demonstrate an atheroprotective role for TLR4 in response to infection with the oral pathogen P. gingivalis. Our results point to a role for pathogen-specific TLR signaling in chronic inflammation and atherosclerosis.

Retinaldehyde represses adipogenesis and diet-induced obesity.

The metabolism of vitamin A and the diverse effects of its metabolites are tightly controlled by distinct retinoid-generating enzymes, retinoid-binding proteins and retinoid-activated nuclear receptors. Retinoic acid regulates differentiation and metabolism by activating the retinoic acid receptor and retinoid X receptor (RXR), indirectly influencing RXR heterodimeric partners. Retinoic acid is formed solely from retinaldehyde (Rald), which in turn is derived from vitamin A. Rald currently has no defined biologic role outside the eye. Here we show that Rald is present in rodent fat, binds retinol-binding proteins (CRBP1, RBP4), inhibits adipogenesis and suppresses peroxisome proliferator-activated receptor-gamma and RXR responses. In vivo, mice lacking the Rald-catabolizing enzyme retinaldehyde dehydrogenase 1 (Raldh1) resisted diet-induced obesity and insulin resistance and showed increased energy dissipation. In ob/ob mice, administrating Rald or a Raldh inhibitor reduced fat and increased insulin sensitivity. These results identify Rald as a distinct transcriptional regulator of the metabolic responses to a high-fat diet.

Omega-3 fatty acid diglyceride emulsions as a novel injectable acute therapeutic in neonatal hypoxic-ischemic brain injury.

Hypoxic-ischemic encephalopathy (HIE), resulting from a lack of blood flow and oxygen before or during newborn delivery, is a leading cause of cerebral palsy and neurological disability in children. Therapeutic hypothermia (TH), the current standard of care in HIE, is only beneficial in 1 of 7-8 cases. Therefore, there is a critical need for more efficient treatments. We have previously reported that omega-3 (n-3) fatty acids (FA) carried by triglyceride (TG) lipid emulsions provide neuroprotection after experimental hypoxic-ischemic (HI) injury in neonatal mice. Herein, we propose a novel acute therapeutic approach using an n-3 diglyceride (DG) lipid emulsions. Importantly, n-3 DG preparations had much smaller particle size compared to commercially available or lab-made n-3 TG emulsions. We showed that n-3 DG molecules have the advantage of incorporating at substantially higher levels than n-3 TG into an in vitro model of phospholipid membranes. We also observed that n-3 DG after parenteral administration in neonatal mice reaches the bloodstream more rapidly than n-3 TG. Using neonatal HI brain injury models in mice and rats, we found that n-3 DG emulsions provide superior neuroprotection than n-3 TG emulsions or TH in decreasing brain infarct size. Additionally, we found that n-3 DGs attenuate microgliosis and astrogliosis. Thus, n-3 DG emulsions are a superior, promising, and novel therapy for treating HIE.

Fast/Glycolytic muscle fiber growth reduces fat mass and improves metabolic parameters in obese mice.

In contrast to the well-established role of oxidative muscle fibers in regulating whole-body metabolism, little is known about the function of fast/glycolytic muscle fibers in these processes. Here, we generated a skeletal muscle-specific, conditional transgenic mouse expressing a constitutively active form of Akt1. Transgene activation led to muscle hypertrophy due to the growth of type IIb muscle fibers, which was accompanied by an increase in strength. Akt1 transgene induction in diet-induced obese mice led to reductions in body weight and fat mass, resolution of hepatic steatosis, and improved metabolic parameters. Akt1-mediated skeletal muscle growth opposed the effects of a high-fat/high-sucrose diet on transcript expression patterns in the liver and increased hepatic fatty acid oxidation and ketone body production. Our findings indicate that an increase in fast/glycolytic muscle mass can result in the regression of obesity and metabolic improvement through its ability to alter fatty acid oxidation in remote tissues.

Correspondence of fatty acid and drug binding sites on human serum albumin: a two-dimensional nuclear magnetic resonance study.

The ability of human serum albumin (HSA) to bind fatty acids (FA) in multiple sites has been revealed by many studies. Here we detect and characterize nine individual binding sites by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of 18-[(13)C]-oleic acid (OA) complexed with HSA. We characterize site-specific FA binding by addition of (i) different FA molar ratios (from 1:1 to 4:1 OA:HSA) to observe the order of filling and occupancy of binding sites; (ii) methyl-ß-cyclodextrin, as a FA acceptor, to observe the dissociation of FA; and (iii) drugs (with known binding sites in the crystal structure) to reveal the correspondence of three NMR peaks with sites in the crystal structure. At 1:1 and 2:1 OA:HSA ratios, three sites were shown to fill sequentially. These high-affinity sites were well resolved from additional sites (one medium-affinity and five low-affinity) observed at 3:1 and 4:1 OA:HSA ratios. Methyl-ß-cyclodextrin extracted OA from individual sites in the reverse order of filling. FA bound in three low-affinity sites were displaced by drugs shown to bind in crystalline HSA to FA sites 7 and 3 (Sudlow's drug sites I and II, respectively) and FA site 6. With this strategy, 2D NMR spectral analysis permits site-specific characterization of the binding of drugs and FA and provides a sensitive probe of the mutual effects of FA and ligand binding.

CD36 enhances fatty acid uptake by increasing the rate of intracellular esterification but not transport across the plasma membrane.

CD36 is a multifunctional protein that enhances cellular fatty acid (FA) uptake, a key step in energy metabolism, and its dysregulation in multiple tissue sites is central to obesity-linked diabetes, a risk factor for atherosclerosis. Although CD36 has been implicated in FA uptake in a correlative way, the molecular mechanisms are not known. Their elucidation in cells is confounded by receptor-mediated uptake of low-density lipoprotein by CD36 and the competitive and/or contributive effects of other proteins involved in FA transport and metabolism, which include caveolin(s), fatty acid transport protein (FATP), intracellular fatty acid binding protein, and enzymes involved in the conversion of FAs to esters. Here we utilized a simpler cellular system (HEK cells), which lack caveolin-1, CD36, and FATP and metabolize FAs slowly compared to the time frame of transmembrane FA movement. Our previous studies of HEK cells showed that caveolin-1 affects FA binding and translocation across the plasma membrane and but not FA esterification [Simard, J. R., et al. (2010) J. Lipid Res. 51 (5), 914-922]. Our key new finding is that CD36 accelerates FA uptake and extensive incorporation into triglycerides, a process that is slower (minutes) than transmembrane movement (seconds). Real-time fluorescence measurements showed that the rates of binding and transport of oleic acid into cells with and without CD36 were not different. Thus, CD36 enhances intracellular metabolism, i.e., esterification, and thereby increases the rate of FA uptake without catalyzing the translocation of FA across the plasma membrane, suggesting that CD36 is central to FA uptake via its effects on intracellular metabolism.

Resolvin E1 heals injured cardiomyocytes: Therapeutic implications and H-FABP as a readout for cardiovascular disease & systemic inflammation.

The purpose of this study is to investigate heart-fatty acid binding protein (H-FABP) leakage from cardiomyocytes as a quantitative measure of cell membrane damage and to test healing by Resolvin E1 (RVE1) as a potential therapeutic for patients with inflammatory diseases (cardiovascular disease and comorbidities) with high morbidity and mortality. Our quantitative ELISA assays demonstrated H-FABP as a sensitive and reliable biomarker for measuring cardiomyocyte damage induced by lipopolysaccharide (LPS) and healing by RvE1, a specialized pro-resolving mediator (SPM) derived from the Omega-3 fatty acid, eicosapentaenoic acid (EPA), a dietary nutrient that balances inflammation to restore homeostasis. RvE1 reduced leakage of H-FABP by up to 86%, which supports our hypothesis that inflammation as a mechanism of injury can be targeted for therapy. H-FABP as a blood biomarker was tested in 40 patients admitted to Boston Medical Center for respiratory distress, (20 patients with and 20 patients without COVID infection). High levels of H-FABP correlated with clinically diagnosed CVD, diabetes, and end-stage renal disease (ESRD) in both patient groups. The level of H-FABP indicates not only CVD damage but is a valuable measure for patients with increased inflammation disease comorbidities.

Solution structure and backbone dynamics of human liver fatty acid binding protein: fatty acid binding revisited.

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical ß-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an "open-cap" conformation or a "swivel-back" mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics.

Effect of disease progression on liver apparent diffusion coefficient and T2 values in a murine model of hepatic fibrosis at 11.7 Tesla MRI.

PURPOSE: To evaluate the effects of hepatic fibrosis on ADC and T(2) values of ex vivo murine liver specimens imaged using 11.7 Tesla (T) MRI. MATERIALS AND METHODS: This animal study was IACUC approved. Seventeen male, C57BL/6 mice were divided into control (n = 2) and experimental groups (n = 15), the latter fed a 3, 5-dicarbethoxy-1, 4-dihydrocollidine (DDC) supplemented diet, inducing hepatic fibrosis. Ex vivo liver specimens were imaged using an 11.7T MRI scanner. Spin-echo pulsed field gradient and multi-echo spin-echo acquisitions were used to generate parametric ADC and T(2) maps, respectively. Degrees of fibrosis were determined by the evaluation of a pathologist as well as digital image analysis. Scatterplot graphs comparing ADC and T(2) to degrees of fibrosis were generated and correlation coefficients were calculated. RESULTS: Strong correlation was found between degrees of hepatic fibrosis and ADC with higher degrees of fibrosis associated with lower hepatic ADC values. Moderate correlation between hepatic fibrosis and T(2) values was seen with higher degrees of fibrosis associated with lower T(2) values. CONCLUSION: Inverse relationships between degrees of fibrosis and both ADC and T(2) are seen, highlighting the utility of these parameters in the ongoing development of an MRI methodology to quantify hepatic fibrosis.

Detection of thrombus size and protein content by ex vivo magnetization transfer and diffusion weighted MRI.

BACKGROUND: To utilize a rabbit model of plaque disruption to assess the accuracy of different magnetic resonance sequences [T1-weighted (T1W), T2-weighted (T2W), magnetization transfer (MT) and diffusion weighting (DW)] at 11.7 T for the ex vivo detection of size and composition of thrombus associated with disrupted plaques. METHODS: Atherosclerosis was induced in the aorta of male New Zealand White rabbits (n = 17) by endothelial denudation and high-cholesterol diet. Subsequently, plaque disruption was induced by pharmacological triggering. Segments of infra-renal aorta were excised fixed in formalin and examined by ex vivo magnetic resonance imaging (MRI) at 11.7 T and histology. RESULTS: MRI at 11.7 T showed that: (i) magnetization transfer contrast (MTC) and diffusion weighted images (DWI) detected thrombus with higher sensitivity compared to T1W and T2W images [sensitivity: MTC = 88.2%, DWI = 76.5%, T1W = 66.6% and T2W = 43.7%, P < 0.001]. Similarly, the contrast-to-noise (CNR) between the thrombus and the underlying plaque was superior on the MTC and DWI images [CNR: MTC = 8.5 ± 1.1, DWI = 6.0 ± 0.8, T1W = 1.8 ± 0.5, T2W = 3.0 ± 1.0, P < 0.001]; (ii) MTC and DWI provided a more accurate detection of thrombus area with histology as the gold-standard [underestimation of 6% (MTC) and 17.6% (DWI) compared to an overestimation of thrombus area of 53.7% and 46.4% on T1W and T2W images, respectively]; (iii) the percent magnetization transfer rate (MTR) correlated with the fibrin (r = 0.73, P = 0.003) and collagen (r = 0.9, P = 0.004) content of the thrombus. CONCLUSIONS: The conspicuity of the thrombus was increased on MTC and DW compared to T1W and T2W images. Changes in the %MTR and apparent diffusion coefficient can be used to identify the organization stage of the thrombus.