Disruption of cholesterol homeostasis triggers periodontal inflammation and alveolar bone loss.

Oral diseases exhibit a significant association with metabolic syndrome, including dyslipidemia. However, direct evidence supporting this relationship is lacking, and the involvement of cholesterol metabolism in the pathogenesis of periodontitis (PD) has yet to be determined. In this study, we showed that high cholesterol caused periodontal inflammation in mice. Cholesterol homeostasis in human gingival fibroblasts was disrupted by enhanced uptake through C-X-C motif chemokine ligand 16 (CXCL16), upregulation of cholesterol hydroxylase (CH25H), and the production of 25-hydroxycholesterol (an oxysterol metabolite of CH25H). Retinoid-related orphan receptor α (RORα) mediated the transcriptional upregulation of inflammatory mediators; consequently, PD pathogenesis mechanisms, including alveolar bone loss, were stimulated. Our collective data provided direct evidence that hyperlipidemia is a risk factor for PD and supported that inhibition of the CXCL16-CH25H-RORα axis is a potential treatment mechanism for PD as a systemic disorder manifestation.

Cell specificity, anti-inflammatory activity, and plausible bactericidal mechanism of designed Trp-rich model antimicrobial peptides.

To develop novel short Trp-rich antimicrobial peptides (AMPs) with potent cell specificity (targeting bacteria but not eukaryotic cells) and anti-inflammatory activity, a series of 11-meric Trp-rich model peptides with different ratios of Leu and Lys/Arg residues, XXWXXWXXWXX-NH(2) (X indicates Leu or Lys/Arg), was synthesized. K(6)L(2)W(3) displayed an approximately 40-fold increase in cell specificity, compared with the natural Trp-rich AMP indolicidin (IN). Lys-containing peptides (K(8)W(3), K(7)LW(3) and K(6)L(2)W(3)) showed approximately 2- to 4-fold higher cell specificities than did their counterparts, the Arg-containing peptides (R(8)W(3), R(7)LW(3) and R(6)L(2)W(3)), indicating that multiple Lys residues are more important than multiple Arg residues in the design of AMPs with good cell specificity. The excellent resistance of d-enantiomers (K(6)L(2)W(3)-D and R(6)L(2)W(3)-D) and Orn/Nle-containing peptides (O(6)L(2)W(3) and O(6)L(2)W(3)) to trypsin digestion compared with the rapid breakdown of the l-enantiomers (K(6)L(2)W(3) and R(6)L(2)W(3)), highlights the clinical potential of such peptides. K(6)L(2)W(3), R(6)L(2)W(3), K(6)L(2)W(3)-D and R(6)L(2)W(3)-D caused weak dye leakage from bacterial membrane-mimicking negatively charged EYPG/EYPE (7:3, v/v) liposomes. Confocal microscopy showed that these peptides penetrated the cell membrane of Escherichia coli and accumulated in the cytoplasm, as observed for buforin-2. Gel retardation studies revealed that the peptides bound more strongly to DNA than did IN. These results suggested that one possible peptide bactericidal mechanism may relate to the inhibition of intracellular functions via interference with DNA/RNA synthesis. Furthermore, some model peptides, containing K(6)L(2)W(3), K(5)L(3)W(3), R(6)L(2)W(3), O(6)L(2)W(3), O(6)L(2)W(3), and K(6)L(2)W(3)-D inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA expression, the release of nitric oxide (NO) following LPS stimulation in RAW264.7 cells and had powerful LPS binding activities at bactericidal concentrations. Collectively, our results indicated that these peptides have potential for future development as novel antimicrobial and anti-inflammatory agents.

NAMPT enzyme activity regulates catabolic gene expression in gingival fibroblasts during periodontitis.

Periodontal disease is one of the most prevalent chronic disorders worldwide. It is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss. Here, we focused on the role of adipokines, which are locally expressed by periodontal tissues, in the regulation of catabolic gene expression leading to periodontal inflammation. The expression of the nicotinamide phosphoribosyltransferase (NAMPT) adipokine was dramatically increased in inflamed human and mouse gingival tissues. NAMPT expression was also increased in lipopolysaccharide- and proinflammatory cytokine-stimulated primary cultured human gingival fibroblasts (GF). Adenovirus-mediated NAMPT (Ad-Nampt) overexpression upregulated the expression and activity of COX-2, MMP1 and MMP3 in human GF. The upregulation of IL-1ß- or Ad-Nampt-induced catabolic factors was significantly abrogated by the intracellular NAMPT (iNAMPT) inhibitor, FK866 or by the sirtuin (SIRT) inhibitor, nicotinamide (NIC). Recombinant NAMPT protein or extracellular NAMPT (eNAMPT) inhibition using a blocking antibody did not alter NAMPT target gene expression levels. Moreover, intragingival Ad-Nampt injection mediated periodontitis-like phenotypes including alveolar bone loss in mice. SIRT2, a part of the SIRT family, was positively associated with NAMPT actions in human GF. Furthermore, in vivo inhibition of the NAMPT-NAD+-SIRT axis by NIC injection in mice ameliorated the periodontal inflammation and alveolar bone erosion caused by intragingival injection of Ad-Nampt. Our findings indicate that NAMPT is highly upregulated in human GF, while its enzymatic activity acts as a crucial mediator of periodontal inflammation and alveolar bone destruction via regulation of COX-2, MMP1, and MMP3 levels.

Bacterial selectivity and plausible mode of antibacterial action of designed Pro-rich short model antimicrobial peptides.

To develop novel Pro-rich model AMPs with shorter length and higher bacterial selectivity/therapeutic index (TI) than natural AMP, indolicidin, we synthesized a series of undodecapeptides derived from the sequence XXPXXPWXPXX-NH2 (X indicates Leu or Lys) with different ratios of Lys and Leu residues. Several Pro-rich model peptides (K7 WP3, K6 WL1 P3, K5 WL2 P3-1, K5 WL2 P3-2, and K4 WL3 P3) had approximate 8- to 11-fold higher bacterial selectivity/TI compared to indolicidin. These peptides selectively bind to negatively charged liposomes (EYPG/EYPG; 7:3, w/w) mimicking bacterial membranes. Their high selectivity to negatively charged phospholipids corresponds well with their high bacterial selectivity. Indolicidin showed almost complete depolarization of the cytoplasmic membrane of Staphylococcus aureus and dye-leakage from negatively charged liposomes at 10 microM, whereas all of Pro-rich model peptides had very little activity in these assays even at 80 microM, as observed in buforin 2. These results suggest that the ultimate target of our designed Pro-rich model peptides is probably the intracellular components (e.g. protein, DNA or RNA) rather than the cytoplasmic membranes. Collectively, our designed Pro-rich short model peptides appear to be excellent candidates for future development as a novel antimicrobial agent.