Electric fields caused by blood flow modulate vascular endothelial electrophysiology and nitric oxide production.

Endothelial cells are exposed to a ubiquitous, yet unexamined electrical force caused by blood flow: the electrokinetic vascular streaming potential (EVSP). In this study, the hypothesis that extremely low frequency (ELF) electric fields parameterized by the EVSP have significant biological effects on endothelial cell properties was studied by measuring membrane potential and nitric oxide production under ELF stimulation between 0 and 2 Hz and 0-6.67 V/m. Using membrane potential and nitric oxide sensitive fluorescent dyes, bovine aortic endothelial cells (BAECs) in culture were studied in the presence and absence of EVSP-modeled electric fields. The transmembrane potential of BAECs was shown to depolarize between 1 and 7 mV with a strong dependency on both the magnitude and frequency of the isolated ELF field. The findings also support a field interaction with a frequency-dependent tuning curve. The ELF field complexly modulates the nitric oxide response to adenosine triphosphate stimulation with potentiation seen with up to a sevenfold increase. This potentiation was also frequency and magnitude dependent. An early logarithmic phase of NO production is enhanced in a field strength-dependent manner, but the ELF field does not modify a later exponential phase. This study shows that using electric fields on the order of those generated by blood flow influences the essential biology of endothelial cells. The inclusion of ELF electric fields in the paradigm of vascular biology may create novel opportunities for advancing both the understanding and therapies for treatment of vascular diseases.

Magnetization transfer magnetic resonance of human atherosclerotic plaques ex vivo detects areas of high protein density.

BACKGROUND: Proteins are major plaque components, and their degradation is related to the plaque instability. We sought to assess the feasibility of magnetization transfer (MT) magnetic resonance (MR) for identifying fibrin and collagen in carotid atherosclerotic plaques ex vivo. METHODS: Human carotid artery specimens (n = 34) were obtained after resection from patients undergoing endarterectomy. MR was completed within 12 hr after surgery on an 11.7T MR microscope prior to fixation. Two sets of T1W spoiled gradient echo images were acquired with and without the application of a saturation pulse set to 10 kHz off resonance. The magnetization transfer ratio (MTR) was calculated, and the degree of MT contrast was correlated with histology. RESULTS: MT with appropriate calibration clearly detected regions with high protein density, which showed a higher MTR (thick fibers (collagen type I) (54 ± 8%)) compared to regions with a low amount of protein including lipid (46 ± 8%) (p = 0.05), thin fibers (collagen type III) (11 ± 6%) (p = 0.03), and calcification (6.8 ± 4%) (p = 0.02). Intraplaque hemorrhage (IPH) with different protein density demonstrated different MT effects. Old (rich in protein debris) and recent IPH (rich in fibrin) had a much higher MTR 69 ± 6% and 55 ± 9%, respectively, compared to fresh IPH (rich in intact red blood cells)(9 ± 3%). CONCLUSIONS: MT MR enhances plaque tissue contrast and identifies the protein-rich regions of carotid artery specimens. The additional information from MTR of IPH may provide important insight into the role of IPH on plaque stability, evolution, and the risk for future ischemic events.

Study of the miscibility of cholesteryl oleate in a matrix of ceramide, cholesterol and fatty acid.

Cholesteryl esters (CE) are not generally abundant but are ubiquitous in living organisms and have markedly different properties from cholesterol because of their acyl chain. The miscibility/immiscibility of CE with biological lipid structures is a key property for their functions. In this work we study the solubility of cholesteryl oleate (ChO) in a model of the stratum corneum lipid matrix composed of ceramide C16, cholesterol and palmitic acid in excess water. Experiments were done in conditions of fully ionized (pH=9.0) and fully neutralized fatty acid (pH=4.0), and differential scanning calorimetry of the ternary mixtures with added ChO at pH=9.0 clearly displayed a main transition with the same maximum temperature, peak shape, and enthalpy, suggesting that ChO was excluded from the remaining lipids. This technique is not conclusive at pH=4.0 because the transitions of the lipid matrix and ChO overlap. The insolubility of ChO at both pH values is supported by X-ray diffraction. Adding the ceramide:cholesterol:fatty acid lipid mixture to ChO did not change the X-ray pattern of the mixture nor that of the ChO. To supplement the above physical techniques, we applied (13)C MAS NMR spectroscopy with C-13 carbonyl-labeled ChO. A single (13)C carbonyl peak from the ChO at 171.5 ppm was observed, indicating exposure to only one environment. The chemical shift was identical to pure ChO below and above the temperature of isotropic liquid formation. Taken together, our results lead to the conclusion that the solubility of ChO is negligible in the ceramide:cholesterol:fatty acid lipid mixture.

In vivo detection of vulnerable atherosclerotic plaque by MRI in a rabbit model.

BACKGROUND: The ability to identify atherosclerotic plaques with a high risk for sudden disruption before stroke or myocardial infarction would be of great utility. We used a rabbit model of controlled atherothrombosis to test whether in vivo MRI can noninvasively distinguish between plaques that disrupt after pharmacological triggering (vulnerable) and those that do not (stable). METHODS AND RESULTS: Atherosclerosis was induced in male New Zealand White (n=17) rabbits by cholesterol diet and endothelial denudation of the abdominal aorta. After baseline (pretrigger) MRI with and without gadolinium contrast, the rabbits underwent 2 pharmacological triggerings to induce atherothrombosis, followed by another MRI 48 hours later (post-triggering). Atherosclerosis was identified by the pretriggered images in all rabbits, and thrombosis was identified in 9 of 17 animals (53%) by post-trigger MRI. After the animals were euthanized, 95 plaques were analyzed; 28 (29.5%) had thrombi (vulnerable) and 67 did not (stable) (70.5%). Pretriggered MRI revealed comparable stenosis in stable and vulnerable plaques, but vulnerable plaques had a larger plaque area (4.8+/-1.6 versus 3.0+/-1.0 mm(2); P=0.01), vessel area (9.2+/-3.0 versus. 15.8+/-4.9 mm(2); P=0.01), and higher remodeling ratio (1.16+/-0.2 versus 0.93+/-0.2; P=0.01) compared with stable plaques. Furthermore, vulnerable plaques more frequently exhibited (1) positive remodeling (67.8% versus 22.3%; P=0.01), in which the plaque is hidden within the vessel wall instead of occluding the lumen; and (2) enhanced gadolinium uptake (78.6% versus 20.9%; P=0.01) associated with histological findings of neovascularization, inflammation, and tissue necrosis. CONCLUSIONS: We demonstrate that in vivo MRI at 3.0 T detects features of vulnerable plaques in an animal model of controlled atherothrombosis. These findings suggest that MRI may be used as a noninvasive modality for localization of plaques that are prone to disruption.

NMR structure of pardaxin, a pore-forming antimicrobial peptide, in lipopolysaccharide micelles: mechanism of outer membrane permeabilization.

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is an important element against permeability of bactericidal agents, including antimicrobial peptides. However, structural determinants of antimicrobial peptides for LPS recognition are not clearly understood. Pardaxins (Pa1, Pa2, Pa3, and Pa4) are a group of pore-forming bactericidal peptides found in the mucous glands of sole fishes. Despite having a low net positive charge, pardaxins contain a broad spectrum of antibacterial activities. To elucidate the structural basis of LPS interactions of pardaxins, herein, we report the first three-dimensional structure of Pa4 bound to LPS micelles. The binding kinetics of Pa4 with LPS is estimated using [(15)N-Leu-19] relaxation dispersion NMR experiments. LPS/Pa4 interactions are further characterized by a number of biophysical methods, including isothermal titration calorimetry, (31)P NMR, saturation transfer difference NMR, dynamic light scattering, and IR spectroscopy. In the LPS-Pa4 complex, Pa4 adopts a unique helix-turn-helix conformation resembling a "horseshoe." Interestingly, the LPS-bound structure of Pa4 shows striking differences with the structures determined in lipid micelles or organic solvents. Saturation transfer difference NMR identifies residues of Pa4 that are intimately associated with LPS micelles. Collectively, our results provide mechanistic insights into the outer membrane permeabilization by pardaxin.

A robust rabbit model of human atherosclerosis and atherothrombosis.

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.

Magnetic resonance microscopy of flows and compressions of the circulatory, respiratory, and digestive systems in pupae of the tobacco hornworm, Manduca sexta.

Circulatory, respiratory, and digestive motions in Manduca sexta pupae were observed using proton-density weighted and fast-imaging with steady-state free procession magnetic resonance microscopy. Proton-density weighted images clearly differentiated pupal air sacs from the hemolymph and organs because, as expected, the air sacs appeared dark in these images. Steady-state free procession imaging allowed real-time monitoring of respiration and circulation, creating movies of hemolymph circulation. Some of the movies show compression and inflation of the air sacs as well as abdominal movements consistent with previously reported ceolopulses. To our knowledge, this is the first magnetic resonance microscopy study of insect circulation and respiration and these preliminary results demonstrate the potential of magnetic resonance microscopy for studying in vivo dynamic processes in insects.

Ex vivo identification of atherosclerotic plaque calcification by a 31P solid-state magnetic resonance imaging technique.

Calcified tissue is a common component of atherosclerotic plaques, and occurs most often in mature plaques. The process of calcification is a poorly understood risk factor that may contribute to a plaque's vulnerability to sudden rupture. In this study a solid-state imaging sequence, termed single-point imaging (SPI), was used to observe calcification directly in ex vivo atherosclerotic plaques. Standards were used to validate the ability of (31)P SPI to detect and differentiate calcification from crystalline cholesterol, phospholipids, and other plaque components. After suitable experimental parameters were found, human carotid specimens obtained by endarterectomy were imaged ex vivo by (31)P solid-state imaging and standard (1)H methods. In contrast to (1)H imaging methods, (31)P imaging detected only the calcification in the plaque.

Structure and orientation of pardaxin determined by NMR experiments in model membranes.

Pardaxins are a class of ichthyotoxic peptides isolated from fish mucous glands. Pardaxins physically interact with cell membranes by forming pores or voltage-gated ion channels that disrupt cellular functions. Here we report the high-resolution structure of synthetic pardaxin Pa4 in sodium dodecylphosphocholine micelles, as determined by (1)H solution NMR spectroscopy. The peptide adopts a bend-helix-bend-helix motif with an angle between the two structure helices of 122 +/- 9 degrees , making this structure substantially different from the one previously determined in organic solvents. In addition, paramagnetic solution NMR experiments on Pa4 in micelles reveal that except for the C terminus, the peptide is not solvent-exposed. These results are complemented by solid-state NMR experiments on Pa4 in lipid bilayers. In particular, (13)C-(15)N rotational echo double-resonance experiments in multilamellar vesicles support the helical conformation of the C-terminal segment, whereas (2)H NMR experiments show that the peptide induces considerable disorder in both the head-groups and the hydrophobic core of the bilayers. These solid-state NMR studies indicate that the C-terminal helix has a transmembrane orientation in DMPC bilayers, whereas in POPC bilayers, this domain is heterogeneously oriented on the lipid surface and undergoes slow motion on the NMR time scale. These new data help explain how the non-covalent interactions of Pa4 with lipid membranes induce a stable secondary structure and provide an atomic view of the membrane insertion process of Pa4.

MSI-78, an analogue of the magainin antimicrobial peptides, disrupts lipid bilayer structure via positive curvature strain.

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.

Membrane composition determines pardaxin's mechanism of lipid bilayer disruption.

Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptide's C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptide's ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition.

An innovative procedure using a sublimable solid to align lipid bilayers for solid-state NMR studies.

Uniaxially aligned phospholipid bilayers are often used as model membranes to obtain structural details of membrane-associated molecules, such as peptides, proteins, drugs, and cholesterol. Well-aligned bilayer samples can be difficult to prepare and no universal procedure has been reported that orients all combinations of membrane-embedded components. In this study, a new method for producing mechanically aligned phospholipid bilayer samples using naphthalene, a sublimable solid, was developed. Using (31)P-NMR spectroscopy, comparison of a conventional method of preparing mechanically aligned samples with the new naphthalene procedure found that the use of naphthalene significantly enhanced the alignment of 3:1 1-palmitoyl-2-oleoyl-phosphatidylethanolamine to 1-palmitoyl-2-oleoyl-phosphatidylglycerol. The utility of the naphthalene procedure is also demonstrated on bilayers of many different compositions, including bilayers containing peptides such as pardaxin and gramicidin. These results show that the naphthalene procedure is a generally applicable method for producing mechanically aligned samples for use in NMR spectroscopy. The increase in bilayer alignment implies that this procedure will improve the sensitivity of solid-state NMR experiments, in particular those techniques that detect low-sensitivity nuclei, such as 15N and 13C.