leupeptins and Arteriosclerosis

The association of periodontitis with atherosclerosis has been suggested from epidemiological studies. Recently, we have reported that macrophages stimulated by Porphyromonas gingivalis formed foam cells in the presence of low-density lipoproteins (LDL). In this study, we examined the direct interactions between LDL and P. gingivalis.. We investigated the aggregation of LDL with P. gingivalis and its outer membrane vesicles (OMVs), degradation of the apo B-100 protein of LDL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, as well as the effects of protease inhibitors or activators on the mobility of LDL by agarose gel shift assays. The binding of P. gingivalis or its OMVs with LDL was demonstrated by western blot analysis. We also examined whether or not the aggregated LDL induced foam cell formation from murine macrophages.. LDL was aggregated in a dose-dependent manner with P. gingivalis and its OMVs. Moreover, degradation of the apo B-100 protein of LDL was directly demonstrated in the presence of P. gingivalis or its OMVs. Furthermore, the gel shift assays indicated that the mobility of LDL was increased in the presence of P. gingivalis. This alteration was attenuated in the presence of the protease inhibitors TLCK and leupeptin and increased in the presence of reducing agents. Moreover, LDL was bound to specific proteins of P. gingivalis suggesting that these proteins may also play a role in aggregation. Finally, the aggregated LDL induced murine macrophages to form foam cells.. These results suggest that P. gingivalis may stimulate foam cell formation, in part, by aggregating LDL by proteolysis of apo B-100.

Topics: Adhesins, Bacterial; Animals; Apolipoprotein B-100; Apolipoproteins B; Arteriosclerosis; Cell Line; Cell Membrane; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Foam Cells; Gingipain Cysteine Endopeptidases; Hemagglutinins; Humans; Leupeptins; Lipoproteins, LDL; Macrophages; Mice; Periodontitis; Porphyromonas gingivalis; Protein Synthesis Inhibitors; Tosyllysine Chloromethyl Ketone; Vacuoles

Lectin-like oxidized LDL receptor-1 (LOX-1) is a type II membrane protein belonging to the C-type lectin family molecules, which can act as a cell-surface endocytosis receptor for atherogenic oxidized LDL. In this study, we show that soluble forms of LOX-1 are present in conditioned media of cultured bovine aortic endothelial cells (BAECs) and CHO-K1 cells stably transfected with LOX-1 cDNA. Immunoblot analysis of conditioned media from TNF-alpha-activated BAECs and CHO-K1 cells stably expressing LOX-1 revealed that soluble LOX-1 has an approximate molecular mass of 35 kDa. In TNF-alpha-activated BAECs, cell-surface expression of LOX-1 precedes soluble LOX-1 production. Cell-surface biotinylation followed by immunoprecipitation and immunoblotting showed that soluble LOX-1 in cell-conditioned media is derived from LOX-1 expressed on the cell surface. Production of soluble LOX-1 was inhibited by PMSF, suggesting that PMSF-sensitive proteases may be involved in this process. Purification of soluble LOX-1 by high-performance liquid chromatography and N-terminal amino acid sequencing of soluble LOX-1 identified the 2 cleavage sites between Arg(86)-Ser(87) and Lys(89)-Ser(90), which were located in the membrane proximal extracellular domain of LOX-1. The data demonstrate that cell-surface LOX-1 can be cleaved at 2 different sites and transformed into soluble forms. Further studies may explore therapeutic and diagnostic applications of soluble LOX-1 in atherosclerotic diseases.

Topics: Amino Acid Sequence; Animals; Aorta; Aprotinin; Arteriosclerosis; Biotinylation; Cattle; Cell Membrane; CHO Cells; Cricetinae; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Endopeptidases; Endothelium, Vascular; Glycoproteins; Lectins; Leupeptins; Lipoproteins, LDL; Membrane Proteins; Molecular Sequence Data; Pepstatins; Protease Inhibitors; Protein Structure, Tertiary; Receptors, LDL; Receptors, Oxidized LDL; Serine Proteinase Inhibitors; Solubility; Tosyl Compounds; Tosyllysine Chloromethyl Ketone; Tumor Necrosis Factor-alpha