Porphyromonas gingivalis infection alters Nrf2-phase II enzymes and nitric oxide in primary human aortic endothelial cells.

BACKGROUND: Periodontal disease (PD) is known to be associated with endothelial dysfunction in patients with coronary artery and/or cardiovascular disease. In our study, we sought to explore the virulence of P. gingivalis (Pg) affecting glycogen synthase kinase 3 beta (GSK-3ß)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/tetrahydrobiopterin (BH4 )/ nitric oxide synthase (NOS) expression in primary human aortic endothelial cells (pHAECs). METHODS: pHAECs were infected for 48 hours with Pg in vitro using the Human oxygen-Bacteria anaerobic coculture technique. Cell viability was determined, and target gene expression changes were evaluated by quantitative real-time polymerase chain reaction at the end of each incubation period. RESULTS: Pg impaired pHAEC viability 24 hours post-infection. Pg infection reduced mRNA expression levels of endothelial NOS (eNOS), Nrf2, and Phase II enzymes (heme oxygenase-1, catalase, superoxide dismutase-1) in a time-dependent manner. Significant (P <0.05) increase in the inflammatory markers (interleukin [IL]-1ß, IL-6, and tumor necrosis factor-α) were observed in the medium as well as in the infected cells. Interestingly, inducible NOS mRNA levels showed a significant (P <0.05) increase at 12 hours and 24 hours and were reduced at later time points. BH4 (cofactor of eNOS) biosynthesis enzyme dihydrofolate reductase (DHFR, salvage pathway) mRNA levels showed a significant (P <0.05) decrease, while mRNA levels of GSK-3ß were elevated. CONCLUSIONS: These results suggest that periodontal bacterial infection may cause significant changes in the endothelial GSK-3ß/BH4 /eNOS/Nrf2 pathways, which may lead to impaired vascular relaxation. Greater understanding of the factors that adversely affect endothelial cell function could contribute to the development of new therapeutic compounds to treat PD-induced vascular diseases.

Transport of Apolipoprotein B-Containing Lipoproteins through Endothelial Cells Is Associated with Apolipoprotein E-Carrying HDL-Like Particle Formation.

Passage of apolipoprotein B-containing lipoproteins (apoB-LPs), i.e., triglyceride-rich lipoproteins (TRLs), intermediate-density lipoproteins (IDLs), and low-density lipoproteins (LDLs), through the endothelial monolayer occurs in normal and atherosclerotic arteries. Among these lipoproteins, TRLs and IDLs are apoE-rich apoB-LPs (E/B-LPs). Recycling of TRL-associated apoE has been shown to form apoE-carrying high-density lipoprotein (HDL)-like (HDLE) particles in many types of cells. The current report studied the formation of HDLE particles by transcytosis of apoB-LPs through mouse aortic endothelial cells (MAECs). Our data indicated that passage of radiolabeled apoB-LPs, rich or poor in apoE, through the MAEC monolayer is inhibited by filipin and unlabeled competitor lipoproteins, suggesting that MAECs transport apoB-LPs via a caveolae-mediated pathway. The cholesterol and apoE in the cell-untreated E/B-LPs, TRLs, IDLs, and LDLs distributed primarily in the low-density (LD) fractions (d ≤ 1.063). A substantial portion of the cholesterol and apoE that passed through the MAEC monolayer was allotted into the high-density (HD) (d > 1.063) fractions. In contrast, apoB was detectable only in the LD fractions before or after apoB-LPs were incubated with the MAEC monolayer, suggesting that apoB-LPs pass through the MAEC monolayer in the forms of apoB-containing LD particles and apoE-containing HD particles.

Overexpression of Catalase Enhances Benzo(a)pyrene Detoxification in Endothelial Microsomes.

We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of NAD(P)H: quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1µM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites, and enhances the ratio of BaP phenolic metabolites versus diol/diones in endothelial microsomes.

Akt isoform-dependent regulation of ATP-Binding cassette A1 expression by apolipoprotein E.

We previously reported that apolipoprotein E (apoE) upregulates ATP-binding cassette transporter A1 (ABCA1) transcription through phosphatidylinositol 3-kinase (PI3K). Here we demonstrate that treatment of murine macrophages with human apoE3 enhanced Akt phosphorylation, and upregulated ABCA1 protein and mRNA expression. Inhibition of PI3K weakened apoE3-induced Akt phosphorylation, and ABCA1 protein and mRNA increase. In contrast, inhibition of Akt only diminished apoE-induced ABCA1 protein but not the mRNA level. Suppression of protein synthesis did not erase the ability of apoE3 to increase ABCA1 protein level. Further, apoE3 increased the resistance of ABCA1 protein to calpain-mediated degradation without affecting calpain activity. Treatment of macrophages with apoE3 selectively enhanced the phosphorylation of Akt1 and Akt2, but not Akt3. Knockdown of Akt1 or Akt2 increased and decreased ABCA1 protein level, respectively; while overexpression of these Akt isoenzymes caused changes in ABCA1 protein level opposite to those induced by knockdown of the corresponding Akt. These data imply that apoE3 guards against calpain-mediated ABCA1 degradation through Akt2.

High-density lipoprotein-mediated transcellular cholesterol transport in mouse aortic endothelial cells.

Accumulation of unesterified cholesterol-rich lipid vesicles in the subendothelial space contributes to atherogenesis. Transport of cholesterol from the subendothelial intima back to the circulating blood inhibits atherosclerosis development; however, the mechanism for this process has not been fully defined. Using cultured mouse aortic endothelial cells (MAECs), we observed that unesterified cholesterol can be transported across the endothelial cell monolayer from the basolateral to the apical compartment. Administration of high-density lipoprotein (HDL) or apolipoprotein AI (apoAI) to the apical compartment enhanced transendothelial cholesterol transport in a concentration-dependent manner. Knockdown of ATP-binding cassette transporter G1 (ABCG1) or scavenger receptor class B type I (SR-B1), or inhibition of SR-B1 diminished HDL-induced transendothelial cholesterol transport; while knockdown of ABCA1 reduced apoAI-mediated cholesterol transport. HDL enhanced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt in MAECs. However, inhibition of PI3K or Akt did not reduce HDL-induced transendothelial cholesterol transport. These results suggest that HDL enhances transendothelial cholesterol transport by activation of a mechanism involving ABCA1, ABCG1 and SR-B1 but not involving PI3K and Akt.

A Subregion of Reelin Suppresses Lipoprotein-Induced Cholesterol Accumulation in Macrophages.

Activation of apolipoprotein E receptor-2 (apoER2) and very low density lipoprotein receptor (VLDLR) inhibits foam cell formation. Reelin is a ligand of these receptors. Here we generated two reelin subregions containing the receptor binding domain with or without its C-terminal region (R5-6C and R5-6, respectively) and studied the impact of these peptides on macrophage cholesterol metabolism. We found that both R5-6C and R5-6 can be secreted by cells. Purified R5-6 protein can bind apoER2 and VLDLR. Overexpression of apoER2 in macrophages increased the amount of R5-6 bound to the cell surface. Treatment of macrophages with 0.2 µg/ml R5-6 elevated ATP binding cassette A1 (ABCA1) protein level by ~72% and apoAI-mediated cholesterol efflux by ~39%. In addition, the medium harvested from cells overexpressing R5-6 or R5-6C (R5-6- and R5-6C-conditioned media, respectively) also up-regulated ABCA1 protein expression, which was associated with accelerated cholesterol efflux and enhanced phosphorylation of phosphatidylinositol 3 kinase (PI3K) and specificity protein-1 (Sp1) in macrophages. The increased ABCA1 expression and cholesterol efflux by R5-6- and R5-6C-conditioned media were diminished by Sp1 or PI3K inhibitors mithramycin A and LY294002. Further, the cholesterol accumulation induced by apoB-containing, apoE-free lipoproteins was significantly less in macrophages incubated with R5-6- or R5-6C-conditioned medium than in those incubated with control conditioned medium. Knockdown of apoER2 or VLDLR attenuated the inhibitory role of R5-6-conditioned medium against lipoprotein-induced cholesterol accumulation. These results suggest that the reelin subregion R5-6 can serve as a tool for studying the role of apoER2 and VLDLR in atherogenesis.

Increase in Blood Glutathione and Erythrocyte Proteins Related to Glutathione Generation, Reduction and Utilization in African-American Old Women with Diabetes.

Data from this report demonstrate that the plasma and erythrocyte levels of total glutathione (TGSH) are significantly lower in nondiabetic old women than in their young counterparts, and significantly higher in diabetic patients than in age-matched nondiabetic controls. The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) declines with age and diabetes, and shows an order as follows: nondiabetic young > nondiabetic old > diabetic old women. In addition, advanced glycation end-products (AGEs) accumulates in RBCs obtained from diabetic patients but not in those from young and old nondiabetic controls. The erythrocyte levels of glutamate cysteine ligase catalytic subunit (GCLC), glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR), glutathione peroxidase-1 (GPx1), glutathione S-transferase-ρ1 (GST-ρ1) and glyoxalase I (Glo1) are comparable in nondiabetic young and old women, but significantly higher in diabetic patients than in age-matched nondiabetic controls. Oxidative stress has been suggested to upregulate the expression of these proteins. It is possible that increase in oxidative stress in diabetes, reflected by reduced GSH/GSSG ratio and accumulation of AGEs, upregulates the expression of proteins involved in glutathione synthesis, reduction and utilization in erythrocyte precursor cells, and that overexpression of GCLC is, at least partially, responsible for the increased TGSH in diabetes.

Up-regulation of cholesterol absorption is a mechanism for cholecystokinin-induced hypercholesterolemia.

Excessive absorption of intestinal cholesterol is a risk factor for atherosclerosis. This report examines the effect of cholecystokinin (CCK) on plasma cholesterol level and intestinal cholesterol absorption using the in vivo models of C57BL/6 wild-type and low density lipoprotein receptor knock-out (LDLR(-/-)) mice. These data were supported by in vitro studies involving mouse primary intestinal epithelial cells and human Caco-2 cells; both express CCK receptor 1 and 2 (CCK1R and CCK2R). We found that intravenous injection of [Thr(28),Nle(31)]CCK increased plasma cholesterol levels and intestinal cholesterol absorption in both wild-type and LDLR(-/-) mice. Treatment of mouse primary intestinal epithelial cells with [Thr(28),Nle(31)]CCK increased cholesterol absorption, whereas selective inhibition of CCK1R and CCK2R with antagonists attenuated CCK-induced cholesterol absorption. In Caco-2 cells, CCK enhanced CCK1R/CCK2R heterodimerization. Knockdown of both CCK1R and CCK2 or either one of them diminished CCK-induced cholesterol absorption to the same extent. CCK also increased cell surface-associated NPC1L1 (Niemann-Pick C1-like 1) transporters but did not alter their total protein expression. Inhibition or knockdown of NPC1L1 attenuated CCK-induced cholesterol absorption. CCK enhanced phosphatidylinositide 3-kinase (PI3K) and Akt phosphorylation and augmented the interaction between NPC1L1 and Rab11a (Rab-GTPase-11a), whereas knockdown of CCK receptors or inhibition of G protein ßγ dimer (Gßγ) diminished CCK-induced PI3K and Akt phosphorylation. Inhibition of PI3K and Akt or knockdown of PI3K diminished CCK-induced NPC1L1-Rab11a interaction and cholesterol absorption. Knockdown of Rab11a suppressed CCK-induced NPC1L1 translocation and cholesterol absorption. These data imply that CCK enhances cholesterol absorption by activation of a pathway involving CCK1R/CCK2R, Gßγ, PI3K, Akt, Rab11a, and NPC1L.

Cholecystokinin elevates mouse plasma lipids.

Cholecystokinin (CCK) is a peptide hormone that induces bile release into the intestinal lumen which in turn aids in fat digestion and absorption in the intestine. While excretion of bile acids and cholesterol into the feces eliminates cholesterol from the body, this report examined the effect of CCK on increasing plasma cholesterol and triglycerides in mice. Our data demonstrated that intravenous injection of [Thr28, Nle31]-CCK at a dose of 50 ng/kg significantly increased plasma triglyceride and cholesterol levels by 22 and 31%, respectively, in fasting low-density lipoprotein receptor knockout (LDLR(-/-)) mice. The same dose of [Thr28, Nle31]-CCK induced 6 and 13% increases in plasma triglyceride and cholesterol, respectively, in wild-type mice. However, these particular before and after CCK treatment values did not achieve statistical significance. Oral feeding of olive oil further elevated plasma triglycerides, but did not alter plasma cholesterol levels in CCK-treated mice. The increased plasma cholesterol in CCK-treated mice was distributed in very-low, low and high density lipoproteins (VLDL, LDL and HDL) with less of an increase in HDL. Correspondingly, the plasma apolipoprotein (apo) B48, B100, apoE and apoAI levels were significantly higher in the CCK-treated mice than in untreated control mice. Ligation of the bile duct, blocking CCK receptors with proglumide or inhibition of Niemann-Pick C1 Like 1 transporter with ezetimibe reduced the hypercholesterolemic effect of [Thr28, Nle31]-CCK in LDLR(-/-) mice. These findings suggest that CCK-increased plasma cholesterol and triglycerides as a result of the reabsorption of biliary lipids from the intestine.

Up-regulation of ATP binding cassette transporter A1 expression by very low density lipoprotein receptor and apolipoprotein E receptor 2.

Activation of very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (apoER2) results in either pro- or anti-atherogenic effects depending on the ligand. Using reelin and apoE as ligands, we studied the impact of VLDLR- and apoER2-mediated signaling on the expression of ATP binding cassette transporter A1 (ABCA1) and cholesterol efflux using RAW264.7 cells. Treatment of these mouse macrophages with reelin or human apoE3 significantly increased ABCA1 mRNA and protein levels, and apoAI-mediated cholesterol efflux. In addition, both reelin and apoE3 significantly increased phosphorylated disabled-1 (Dab1), phosphatidylinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ), and specificity protein 1 (Sp1). This reelin- or apoER2-mediated up-regulation of ABCA1 expression was suppressed by 1) knockdown of Dab1, VLDLR, and apoER2 with small interfering RNAs (siRNAs), 2) inhibition of PI3K and PKC with kinase inhibitors, 3) overexpression of kinase-dead PKCζ, and 4) inhibition of Sp1 DNA binding with mithramycin A. Activation of the Dab1-PI3K signaling pathway has been implicated in VLDLR- and apoER2-mediated cellular functions, whereas the PI3K-PKCζ-Sp1 signaling cascade has been implicated in the regulation of ABCA1 expression induced by apoE/apoB-carrying lipoproteins. Taken together, these data support a model in which activation of VLDLR and apoER2 by reelin and apoE induces ABCA1 expression and cholesterol efflux via a Dab1-PI3K-PKCζ-Sp1 signaling cascade.

A novel function of apolipoprotein E: upregulation of ATP-binding cassette transporter A1 expression.

Despite the well known importance of apolipoprotein (Apo) E in cholesterol efflux, the effect of ApoE on the expression of ATP-binding cassette transporter A1 (ABCA1) has never been investigated. The objective of this study was to determine the effect of ApoE on ApoB-carrying lipoprotein-induced expression of ABCA1, a protein that mediates cholesterol efflux. Our data demonstrate that ApoB-carrying lipoproteins obtained from both wild-type and ApoE knockout mice induced ApoAI-mediated cholesterol efflux in mouse macrophages, which was associated with an enhanced ABCA1 promoter activity, and an increased ABCA1 mRNA and protein expression. In addition, these lipoproteins increased the level of phosphorylated specificity protein 1 (Sp1) and the amount of Sp1 bound to the ABCA1 promoter. However, all these inductions were significantly diminished in cells treated with ApoE-free lipoproteins, when compared to those treated with wild-type lipoproteins. Enrichment with human ApoE3 reversed the reduced inducibility of ApoE-free lipoproteins. Moreover, we observed that inhibition of Sp1 DNA-binding by mithramycin A diminished ABCA1 expression and ApoAI-mediated cholesterol efflux induced by ApoB-carrying lipoproteins, and that mutation of the Sp1-binding motif in the ABCA1 promoter region diminished ApoB-carrying lipoprotein-induced ABCA1 promoter activity. Collectively, these data suggest that ApoE associated with ApoB-carrying lipoproteins has an upregulatory role on ABCA1 expression, and that induction of Sp1 phosphorylation is a mechanism by which ApoE upregulates ABCA1 expression.

Transcriptional regulation of ATP-binding cassette transporter A1 expression by a novel signaling pathway.

ATP-binding cassette transporter A1 (ABCA1) is a membrane-bound protein that regulates the efflux of cholesterol derived from internalized lipoproteins. Using a mouse macrophage cell line, this report studied the impact of low-density lipoproteins (LDL) on ABCA1 expression and the signaling pathway responsible for lipoprotein-induced ABCA1 expression. Our data demonstrated that treatment of macrophages with LDL increased ABCA1 mRNA and protein levels 4.3- and 3.5-fold, respectively. LDL also induced an ∼2-fold increase in macrophage surface expression of ABCA1 and a 14-fold-increase in apolipoprotein AI-mediated cholesterol efflux. In addition, LDL significantly increased the level of phosphorylated specificity protein 1 (Sp1) and the amount of Sp1 bound to the ABCA1 promoter without alteration in total Sp1 protein level. Mutation of the Sp1 binding site in the ABCA1 promoter and inhibition of Sp1 DNA binding with mithramycin A suppressed the ABCA1 promoter activity and reduced the ABCA1 expression level induced by LDL. LDL treatment also elevated protein kinase C-ζ (PKC-ζ) phosphorylation and induced PKC-ζ binding with Sp1. Inhibition of PKC-ζ with kinase inhibitors or overexpression of kinase-dead PKC-ζ attenuated Sp1 phosphorylation and ABCA1 expression induced by LDL. These results demonstrate for the first time that activation of the PKCζ-Sp1 signaling cascade is a mechanism for regulation of LDL-induced ABCA1 expression.

Apolipoprotein E-Deficient Lipoproteins Induce Foam Cell Formation by Activation of PERK-EIF-2α Signaling Cascade.

Transformation of macrophages into foam cells by apolipoprotein (Apo) E-deficient, ApoB48-containing (E(-)/B48) lipoproteins has been shown to be associated with increased phosphorylation of eukaryotic initiation factor-2α (eIF-2α). The present report examined the causal relationship between eIF-2α phosphorylation and lipid accumulation in macrophages induced by E(-)/B48 lipoproteins. E(-)/B48 lipoproteins increased eIF-2α phosphorylation and cholesterol ester accumulation, while lipoprotein degradation decreased and lysosomal acid lipase and cathepsin B mRNA translation was inhibited in mouse peritoneal macrophages (MPMs). These responses were overcome by overexpression of a nonphosphorylatable eIF-2α mutant in MPMs. Incubation of MPMs with E(-)/B48 lipoproteins also increased the phosphorylation of RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK), but not other eIF-2α kinases. Overexpression of a nonphosphorylatable PERK mutant inhibited PERK and eIF-2α phosphorylation, and alleviated cholesterol ester accumulation induced by E(-)/B48 lipoproteins. PERK is an eIF-2α kinase activated by endoplasmic reticulum (ER) stress. Taken together, findings from this report suggest that induction of ER stress, i.e., activation of the PERK-eIF2α signaling cascade, is a mechanism by which E(-)/B48 lipoproteins down-regulate lysosomal hydrolase synthesis, inhibit lysosomal lipoprotein degradation, and increase intracellular lipoprotein and cholesterol ester accumulation, resulting in foam cell formation.