Porphyromonas Gingivalis Lipopolysacchride Altered Atherosclerotic Gene Profiling In Oxidized Low Density Lipoprotein Induced Foam Cells

Current epidemiological data demonstrate that there is a positive correlation between periodontitis and cardiovascular diseases, meanwhile blood infusion of Porphyromonas gingivalis (P. gingivalis)lipopolysaccharides(LPS) leaded to the exacerbation of atherosclerosis in apoE-/- mice. Foam cells formation induced by modified low density lipoprotein from macrophages was the key event in atherogenesis. The purpose of the study was to evaluated the effect of P. gingivalis-LPS on atherosclerotic gene expression during foam cells formation and after its formation, thus to unravel the mechanism of P. gingivalis-LPS to induce atherogenesis.Part 1 Construction of foam cells model from human macrophages in vitroPurpose: To construct foam cells model from human macrophages in vitro.Methods: THP-1 human monocyte line was stimulated with 160nmo1/L PMA for 36h to induce macrophages, then different concentrations oxLDL were added, after 36h and 48h, cellular morphological evaluation and cholesterol estate(CE) detection by biochemistry were utilized to evaluate foam cells formation.Results: oxLDL induction for 48h leaded to the percentage of CE to total cholesterol (TC) exceeding 60%, with lipid particles in macrophage as shown in Oil-red staining. Conclusions: Macrophages from PMA preincubated THP-1 monocytes could be successfully induced into foam cells by oxLDL in vitro.Part 2 Effect of P.gingivalis-LPS on atherosclerotic genetic profiling and cytokine expression during foam cells formation Purpose: To examine the effect of P.gingivalis-LPS on atherosclerotic genes and cytokines expression during foam cells formation.Methods:1. Levels of IL-1βand TNF-αin RMPI1640,oxLDL and oxLDL+P.gingivalis-LPS treated macrophage were determined by enzyme-linked immunosorbent assay (ELISA).2. NF-κBp65 translocation after adding of stimulants were detected by cytoimmunochemistry.3. IκB-αlevels in cytosol were evaluated with Western blot.4. Transcription of IL-1β, IL-10, IL-12 P40 and IL-18 2h after stimulation were examined by Real time PCR.5. An atherosclerotic gene microarray was utilized to detect the expression of 84 genes 2h after stimulation.Results:1. Both oxLDL and P.gingivalis-LPS could enhance IL-1βand TNF-αsecretion, but an additive effect was not observed.2. Both oxLDL and P.gingivalis-LPS could increase NF-κB p65 translocation, and an additive effect was found.3. oxLDL could down-regulate IκB-αlevel in cytosol, and P.gingivalis-LPS could further decrease its expression.4. oxLDL could stimulate the transcription of IL-1β, IL-10, IL-12 P40 and IL-18, P.gingivalis-LPS could enhance such stimulation effect of oxLDL.5. oxLDL alone or with P.gingivalis-LPS could up- or down-regulate more than 40 genes for 2-fold or more. Genes up- or down-regulated by oxLDL mainly became further up-or down-regulated by P.gingivalis-LPS. Adhesion molecules, chemokines, growth factors and apoptotic genes were the genes most influenced by oxLDL or P.gingivalis-LPS.Conclusions: P.gingivalis-LPS could stimulate NF-κB pathway in oxLDL induced foam cell formation and influenced atherogenic genes expression of oxLDL during foam cell formation.Part 3 Effects of P. gingivalis-LPS on atherogenic genes expression in foam cellsPurpose: To evaluate the influence of P.gingivalis-LPS on atherosclerotic genes expression in foam cells.Methods: P.gingivalis-LPS were added into foam cells from oxLDL stimulated macrophages. IκB-αlevel in cytosol were dertermined by Western blot. Real-time PCR was used to detect IL-1β, IL-10, IL-12 P40 and IL-18 mRNA , and microarray was utilized to evaluate atherosclerotic genes expression in foam cells. Results: P.gingivalis-LPS could decrease IκB-αlevel in foam cells, enhance IL-1βand IL-12P40 transcription and up-regulate more than 10 genes in foam cells.Part 4 Effects of P.gingivalis-LPS on apoptosis in oxLDL induced macrophagesPurpose: To investigate the effect of P.gingivalis-LPS on apoptosis in oxLDL induced macrophages.Methods: Macrophage growth and proliferation were examined using MTT method. Apoptosis was evaluated by AO/EB staining. mRNA of p53, caspase-3 and c-Myc were detected with Real-time PCR, gene microarray was utilized to examine more ten apoptotic genes expression in macrophages.Results: oxLDL could enhance macrophage proliferation, and P.gingivalis-LPS had a cytotoxic effect to inhibit growth and proliferation of macrophages. Apoptosis could be examined in all groups by AO/EB staining. P.gingivalis-LPS could inhibit c-Myc expression, antagonize the inhibition of oxLDL on caspase-3 and p53 during foam cell formation, meawhile P.gingivalis-LPS could enhance caspase-3 expression and decrease p53 transcription in foam cells, and oxLDL and P.gingivalis-LPS influence a major part of apoptotic gene expression in microarray.Conclusions: oxLDL and P.gingivalis-LPS influenced apoptotic gene expression in macrophages, which might play an important role in atherogenesis.