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BackgroundThe blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood.Methodology/Principal FindingsIn this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor.Conclusions/SignificanceThis study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

We analyze the crystallization and collapse transition of a simple model for flexible polymer chains on simple-cubic and face-centered-cubic lattices by means of sophisticated chain-growth methods. In contrast to the bond-fluctuation polymer model in certain parameter ranges, where these two conformational transitions were found to merge in the thermodynamic limit, we conclude from our results that the two transitions remain well separated in the limit of infinite chain lengths. The reason for this qualitatively distinct behavior is presumably due to the ultrashort attractive interaction range in the lattice models considered here.

We introduce a parallel Wang-Landau method based on the replica-exchange framework for Monte Carlo simulations. To demonstrate its advantages and general applicability for simulations of complex systems, we apply it to different spin models including spin glasses, the Ising model, and the Potts model, lattice protein adsorption, and the self-assembly process in amphiphilic solutions. Without loss of accuracy, the method gives significant speed-up and potentially scales up to petaflop machines.

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Thrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence-Trp-Ser-Pro-Trp- and a recombinant heparin binding fragment from the amino-terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin-binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin-dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin-binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time-dependent and reversible. Thus, the antiproliferative activities of the thrombospondin peptide and recombinant heparin-binding domain result at least in part from competition with heparin-dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp-Ser-Xaa-Trp sequences in the type I repeats and the amino-terminal domain play roles in the antiproliferative activity of thrombospondin.

Peptides from the three type I repeats of human endothelial cell thrombospondin, containing the consensus sequence -Trp-Ser-Xaa-Trp-, bind to sulfated glycoconjugates including heparin and sulfatide. The peptides are potent inhibitors for the binding of thrombospondin, laminin, or apolipoprotein E to these ligands. The thrombospondin peptides that inhibit heparin binding, but not adjacent peptides from the thrombospondin sequence containing the previously identified adhesive motif Val-Thr-Cys-Gly, promote melanoma cell adhesion when immobilized on plastic. Melanoma cell adhesion to the immobilized peptides is inhibited by soluble recombinant heparin-binding fragment of thrombospondin. The peptides also inhibit heparin-dependent binding of thrombospondin or laminin to human melanoma cells. The active peptides lack any previously identified heparin-binding consensus sequences and most do not contain any basic amino acids. Studies with homologous peptides showed that the tryptophan residues are required for binding. Adjacent basic residues in the second type I repeat enhance binding to heparin but not to sulfatide. Thus the type I peptides of thrombospondin define a distinct class of heparin-binding peptides.

An important feature of Alzheimer's disease (AD) is the cerebral deposition of amyloid. The main component of the amyloid is a 39-44-amino acid residue protein called amyloid beta (A beta), which also exists as a normal protein in biological fluids, known as soluble A beta. A major risk factor for late-onset AD is the inheritance of the apolipoprotein (apo) E4 isotype of apoE. How apoE is involved in the pathogenesis of AD is unclear; however, evidence exists for a direct apoE/A beta interaction. We and others have shown that apoE copurifies with A beta from AD amyloid plaques and that under certain in vitro conditions apoE promotes a beta-sheet structure in A beta peptides. Currently we document the high affinity binding of A beta peptides to both human recombinant apoE3 and -E4 with a KD of 20 nM. This interaction is greatly influenced by the conformational state of the A beta peptide used. Furthermore, we show that the fibril modulating effect of apoE is also influenced by the initial secondary structure of the A beta peptide. The preferential binding of apoE to A beta peptides with a beta-sheet conformation can in part explain the copurification of A beta and apoE from AD amyloid plaques.