Spectroscopic photoacoustic imaging has the potential to discriminate between normal and lipid-rich atheromatous areas of arterial tissue by exploiting the differences in the absorption spectra of lipids and normal arterial tissue in the 740 to 1400 nm wavelength range. Identification of regions of high lipid concentration would be useful to identify plaques that are likely to rupture (vulnerable plaques). To demonstrate the feasibility of visualizing lipid-rich plaques, samples of human aortas were imaged in forward mode, at wavelengths of 970 and 1210 nm. It was shown that the structure of the arterial wall and the boundaries of lipid-rich plaques obtained from the photoacoustic images were in good agreement with histology. The presence of lipids was also confirmed by comparing the photoacoustic spectra (740 to 1400 nm) obtained in a region within the plaque to the spectral signature of lipids. Furthermore, a lipid-rich plaque was successfully imaged while illuminating the sample through 2.8 mm of blood demonstrating the possibility of implementing the photoacoustic technique in vivo.

We have previously reported that plasma apolipoprotein (apo) E-containing high density lipoprotein particles have a potent anti-platelet action, apparently by occupying saturable binding sites in the cell surface. Here we show that purified apoE (10 -50 g/ml), complexed with phospholipid vesicles (dimyristoylphosphatidylcholine, DMPC), suppresses platelet aggregation induced by ADP, epinephrine, or collagen. This effect was not due to sequestration of cholesterol from platelet membranes; apoE⅐DMPC chemically modified with cyclohexanedione (cyclohexanedione-apoE⅐DMPC) did not inhibit aggregation but nevertheless removed similar amounts of cholesterol as untreated complexes, about 2% during the aggregation period. Rather we found that apoE influenced intracellular platelet signaling. Thus, apoE⅐DMPC markedly increased cGMP in ADP-stimulated platelets which correlated with the resulting inhibition of aggregation (r ‫؍‬ 0.85; p < 0.01, n ‫؍‬ 10), whereas cyclohexanedione-apoE⅐DMPC vesicles had no effect. One important cellular mechanism for up-regulation of cGMP is through stimulation of nitric oxide (NO) synthase, the NO generated by conversion of Larginine to L-citrulline, binds to and activates guanylate cyclase. This signal transduction pathway was implicated by the finding that NO synthase inhibitors of distinct structural and functional types all reversed the anti-platelet action of apoE, whereas a selective inhibitor of soluble guanylate cyclase, 1H- platelets) than controls (0.18 ؎ 0.03; p < 0.05). In addition, hemoglobin which avidly binds NO also suppressed the anti-aggregatory effect, indicating that apoE stimulated sufficient production of NO by platelets for extracellular release to occur. We conclude that apoE inhibits platelet aggregation through the L-arginine:NO signal transduction pathway. Human apolipoprotein E (apoE)1 is a 299-residue protein of molecular mass 34 kDa found in the surface of circulating triglyceride-rich lipoproteins (very low density lipoprotein and chylomicrons, or their remnants) and certain HDL particles (1). Its major function is to mediate hepatic clearance of lipoproteins through interaction with two receptors, the low density lipoprotein or B,E receptor and an apoE-specific receptor, most probably the low density lipoprotein receptor-related protein (2). When the apoE polypeptide is dysfunctional or absent severe hyperlipidemia and atherosclerosis in humans or animal models ensues (1, 3-5). Although apoE is synthesized predominantly by the liver, macrophages also secrete apoE; this appears important for facilitating local cholesterol redistribution, for reverse cholesterol transport, and for restricting development of atherosclerotic lesions (6). Indeed, atherosclerosis in apoE-deficient (apoE Ϫ/Ϫ ) mice can be prevented by transplantation of normal murine bone marrow cells (5), by macrophage-specific expression of the human apoE transgene (7), or by adenovirus-mediated gene replacement (8).Recently, we proposed an additional anti-atherogenic role for apoE. We found that H...

Hepatitis C virus (HCV) enters cells via a pH-and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BIHepatitis C virus (HCV) is an enveloped positive-strand RNA virus and the sole member of the genus Hepacivirus, within the Flaviviridae. Approximately 170 million individuals are infected with HCV worldwide, and the majority are at risk of developing serious progressive liver disease. The principal reservoir for viral replication is believed to be hepatocytes within the liver, and until recently, minimal information was available on the mechanism(s) of HCV entry. However, the last 3 years have seen several advances that contribute to our ability to study HCV hepatotropism. First, the development of the retrovirus pseudoparticle system, in which cell entry is dependent upon the expression of HCV glycoproteins (HCVpp) (4, 20), and secondly, the ability of the JFH strain of HCV to release infectious particles in cell culture (HCVcc) (25,51,55).Early studies with a truncated soluble version(s) of HCV E2 (sE2) allowed the identification of a number of interacting cellular proteins, including the tetraspanin CD81 (16, 37), scavenger receptor class B type I (SR-BI) (43), and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) and the related molecule DC-SIGN(R), or L-SIGN (15,18,27,40). The availability of HCVpp and infectious HCVcc has provided tools for validating these receptor candidates.CD81 is a nonglycosylated member of the tetraspanin family of proteins. Both HCVpp and HCVcc infectivities are inhibited by soluble forms of CD81 and by anti-CD81 monoclonal antibodies (MAbs), suggesting that CD81 is required for HCV infection (6,20,25). Definitive experiments showing that expression of CD81 in a CD81-negative human liver cell line, HepG2, confers infectivity support a critical role of CD81 in HCV cell entry (24,25,54,55).SR-BI is expressed within the liver, steroidogenic tissue, and macrophages and is considered to be the major receptor for high-density lipoprotein (HDL) (23). SR-BI mediates the traffic of cholesterol to and from lipoproteins by selective cholesterol uptake, cholesterol efflux, and receptor-mediated endocytosis (1,34,42,44). The SR-BI gene gives rise to at least two mRNA splice variants. The SR-BII isoform differs from SR-BI at the C terminus, which is reported to confer intracellular localization on 33,52).Experiments to validate the role of SR-BI in HCV infection have proven difficult, since all cell types studied to date express SR-BI, and small interfering RNA silencing has a modest effect on HCVpp infectivity (6,24,48). The native lipoprotein ligands have differential effects on HCV infectivity: HDL enhances infectivity, low-density (LDL) and very low-density lipoproteins (VLDL) have no effect (5, 48), and oxidized LDL abrogates infectivity (50), suggesting a complex interplay between SR-BI, lipoproteins, and HCV. Treatment of target cells

Incubation of normal plasma low-density lipoprotein (LDL) with erythrocytes results in echinocyte formation; the effect is attributed to stimulation of spectrin dephosphorylation through binding of LDL to the cell surface (Hui & Harmony, 1979, Biochimica et Biophysica Acta, 550, 407). No shape change occurs when erythrocytes are incubated with normal highdensity lipoprotein (HDL) and LDL-induced echinocyte formation is inhibited by HDL. We have established that as a consequence of abnormal apoprotein composition (an increased content of ApoE and threonine-poor apoproteins) the HDL of liver disease competes with LD L for binding by the high-affinity receptor on cultured skin fibroblasts. In the present study we have determined whether the abnormal HDL of liver disease (d = 1·063-1·21) will induce echinocyte formation in normal erythrocytes.HDL from several patients which showed the apoprotein abnormalities were tested; all induced marked echinocyte formation when added to a suspension of normal erythrocytes at 37°C (6 x 10 9 cells/ml and 500 ug ofHDL-protein/ml). Patient HDL was 10-100 times more potent than normal LDL and could transform 100% of the cells. Echinocyte formation was rapid (less than 10 s), occurred to a similar extent at 4°C and was also induced by partially delipidated patient HDL. The erythrocyte binding site differed from that on cultured fibroblasts; echinocyte formation was not inhibited by protamine or heparin and also occurred when apoprotein arginine residues were blocked with cyclohexanedione or when erythrocytes from patients with homozygous familial hypercholesterolaemia were used. The morphological changes were associated with increased osmotic resistance and with decreased membrane fluidity as measured by a hydrophobic, fluorescent probe. Attempts to restore biconcave disc morphology by addition of heparin or normal HDL were only moderately successful.Previous studies have implicated changes in erythrocyte membrane lipid composition as underlying the 'spur-cell anaemia' of liver disease (Cooper, Arner, Wiley & Shattil, 1975, Journal of Clinical Investigation, 55, 115); our results suggest that abnormal HDL apoprotein composition may also play a role. DECREASED ER YTHROCYTE MEMBRANE FLUIDITY AND ALTERED LIPID COMPOSITION IN HUMAN LIVER DISEASEAbnormal plasma lipoproteins in patients with liver disease are associated with characteristic changes in erythrocyte membrane lipid composition. The membranes are enriched in cholesterol and lecithin and both the cholesterol/phospholipid (C/P) and lecithin/sphingomyelin (LlSM) molar ratios are increased. Phospholipid fatty acid composition is also abnormal; the proportion of arachidonic acid is decreased and that of palmitic acid raised. In this study we have examined the effects of these membrane lipid abnormalities on membrane fluidity.Erythrocyte membrane fluidity was assessed in 30 patients with a variety of liver diseases, and in 25 normal subjects by using the hydrophobic, fluorescent probe I,6-diphenyl hex a-1,3,5-triene, and the value...