IL-15 aggravates atherosclerotic lesion development in LDL receptor deficient mice.

BACKGROUND: Interleukin 15 (IL-15) is a pro-inflammatory cytokine involved in inflammatory diseases and IL-15 is expressed in atherosclerotic plaques. METHODS: To establish the role of IL-15 in atherosclerosis we studied the effect of IL-15 on atherosclerosis associated cells in vitro and in vivo by neutralizing IL-15 using a DNA vaccination strategy. RESULTS: Upon feeding a Western type diet LDLr(-/-) mice do express higher levels of IL-15 within the spleen and the number of IL-15 expressing cells among blood leukocytes and spleen cells is increased. Addition of IL-15 to macrophages induces the expression TNF-α and CCL-2. After the mice were vaccinated against IL-15, we observe a reduction in plaque size of 75% plaque. Unexpectedly, the relative number of macrophages within the plaque was 2-fold higher in IL-15 vaccinated mice than in control mice. Vaccination against IL-15 leads to an increased cytotoxicity against IL-15 overexpressing target cells, resulting in a reduction in IL-15 expressing cells and macrophages in blood and spleen and a decreased CD4/CD8 ratio. CONCLUSION: Hypercholesterolemia leads to upregulation of IL-15 within spleen and blood. DNA vaccination against IL-15 does markedly reduces atherosclerotic lesion size, but does not promote lesion stability.

Microarray analysis indicates an important role for FABP5 and putative novel FABPs on a Western-type diet.

Liver parenchymal cells play a dominant role in hepatic metabolism and thereby total body cholesterol homeostasis. To gain insight into the specific pathways and genes involved in the response of liver parenchymal cells to increased dietary lipid levels under atherogenic conditions, changes in parenchymal cell gene expression upon feeding a Western-type diet for 0, 2, 4, and 6 weeks were determined using microarray analysis in LDL receptor-deficient mice, an established atherosclerotic animal model. Using ABI Mouse Genome Survey Arrays, we were able to detect 7,507 genes (28% of the total number on an array) that were expressed in parenchymal cells isolated from livers of LDL receptor-deficient mice at every time point investigated. Time-dependent gene expression profiling identified fatty acid binding protein 5 (FABP5) and four novel FABP5-like transcripts located on chromosomes 2, 8, and 18 as important proteins in the primary response of liver parenchymal cells to Western-type diet feeding, because their expression was 16- to 22-fold increased within the first 2 weeks on the Western-type diet. The rapid substantial increase in gene expression suggests that these FABPs may play an important role in the primary protection against the cellular toxicity of cholesterol, free fatty acids, and/or lipid oxidants. Furthermore, as a secondary response to the Western-type diet, liver parenchymal cells of LDL receptor-deficient mice stimulated glycolysis and lipogenesis pathways, resulting in a steady, more atherogenic serum lipoprotein profile (increased VLDL/LDL).

P-selectin as a candidate target in atherosclerosis.

P-selectin is of critical importance in early atherogenesis by initiating leukocyte rolling at the site of endothelial injury. In order to validate P-selectin as a candidate target for the development of anti-atherogenic strategies, we wanted to obtain quantitative information on P-selectin expression, and identify novel peptide-based lead structures that interact with P-selectin. P-selectin mRNA expression in the aortic arch and in other tissues of apoE-deficient (apoE-/-) mice was determined by real-time PCR technology. P-selectin mRNA expression of apoE-/- mice increased steadily with age to levels 14-fold higher than that of control animals. The onset and level of P-selectin expression correlated well with the extent of lesion development, and was more specific for atherosclerotic tissue as compared with other adhesion molecules. Phage display technology was used to obtain novel P-selectin antagonists. Phage display selections resulted in the isolation of a highly P-selectin-specific phage clone. Synthetic peptide-equivalents of this clone displaced the binding of the parent phage and antagonized the binding of a sialyl Lewis(x) analogue to P-selectin. In conclusion, P-selectin expression correlates with early and advanced atherosclerotic lesion development. P-selectin ligands, like the lead structure we have developed here, can therefore be considered as promising tools to identify, target or antagonize P-selectin function within the chronically inflamed arterial wall.

Specific inhibition of P-selectin-mediated cell adhesion by phage display-derived peptide antagonists.

P-selectin is a leukocyte adhesion receptor expressed on activated vascular endothelium and platelets that mediates leukocyte rolling and attachment. Because P-selectin is critically involved in inflammation, we used phage display libraries to identify P-selectin-specific peptides that might interfere with its proinflammatory function. Isolated phage contained a highly conserved amino acid motif. Synthetic peptides showed calcium-dependent binding to P-selectin, with high selectivity over E-selectin and L-selectin. The peptides completely antagonized adhesion of monocyte-derived HL60 cells to P-selectin and increased their rolling velocities in flow chamber experiments. Peptide truncation and alanine-scanning studies indicated that an EWVDV (single-letter amino acid codes) consensus motif sufficed for effective inhibition. Intriguingly, the apparent avidity of the peptides was increased 200-fold when presented in a tetrameric form (2 microM versus 10 nM), which is consistent with the proposed divalent interaction of P-selectin glycoprotein ligand 1 (PSGL-1) with P-selectin. As the EWVDV peptides inhibit the binding of an established glycoside ligand for P-selectin (sulfated Lewis A), it is conceivable that EWVDV interacts with or in close proximity to the actual carbohydrate recognition domain of P-selectin, without being a direct structural mimic of sialyl Lewis(x). These ligands are among the most potent antagonists of P-selectin yet designed. Their high affinity, selectivity, and accessible synthesis provide a promising entry to the development of new anti-inflammatory therapeutics and might be a powerful tool to provide important information on the binding site of P-selectin.

Targeting of peptides to restenotic vascular smooth muscle cells using phage display in vitro and in vivo.

Restenosis after angioplasty occurs in 30-40% of the treated patients. To develop a strategy to deliver drugs to restenotic lesions, we selected phages that bind to proliferating vascular smooth muscle cells (VSMC), from a random constraint 15-mer peptide phage display library. Phages were selected for binding to cultured primary aortic VSMC (in vitro biopanning) and selected for binding to denudated carotid arteries in mice (in vivo biopanning). In vitro biopanning did not result in a consensus sequence, but recurring FLGW and LASR amino acid motifs were identified. In vivo biopanning resulted in two consensus peptides 5G6 (CNIWGVVLSWIGVFPEC) and 5E5 (CESLWGGLMWTIGLSDC). Surprisingly, these two sequences were recovered after both in vitro and in vivo biopanning, but predominantly in vivo. Moreover, a strong recurring motif, IGR, was identified in the in vivo clones. The consensus phages 5G6 and 5E5 bind selectively to VSMC compared to other cell types. Furthermore, they bind preferentially to proliferating VSMC compared to VSMC that were growth arrested, and are effectively internalized by their target cells. The specific binding capacities of 5G6 and 5E5 phages suggest that these peptide sequences can be used for targeting of restenotic lesions, in which proliferating VSMC are the dominant cell type.

The effect of TGF-beta receptor binding peptides on smooth muscle cells.

TGF-beta1 is a potent regulator of vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix (ECM) synthesis. In this study, we selected two peptides, IM-1 and IM-2, that bind to the TGF-beta type II receptor (TGF-beta RII) using phage display. IM-1 and IM-2 bind to the TGF-beta RII, with a K(d) of 1 microM. Like TGF-beta, IM-1 induced VSMC chemotaxis and PAI-1 mRNA expression, as determined using Boyden chambers and real time quantitative PCR. In contrast, IM-2 had no effect on VSMC chemotaxis or PAI-1 induction. Induction of ECM synthesis, involving proteins such as osteopontin and alpha-smooth muscle actin, was determined by ELISA. Osteopontin expression was inhibited by both peptides, but TGF-beta-induced alpha-smooth muscle actin expression could only be inhibited by IM-1. In conclusion, IM-1 activity on VSMC is agonistic with TGF-beta, except for ECM synthesis, whereas the IM-2 peptide is antagonistic for some examined TGF-beta functions.