Foam Cell Targeted Liposomes Co-Encapsulating Superoxide Dismutase and Catalase to Attenuate Atherosclerosis by Inhibiting Oxidative Stress.

BACKGROUND: Oxidative stress, propelled by reactive oxygen species (ROS), serves as a significant catalyst for atherosclerosis (AS), a primary contributor to vascular diseases on a global scale. Antioxidant therapy via nanomedicine has emerged as a pivotal approach in AS treatment. Nonetheless, challenges such as inadequate targeting, subpar biocompatibility, and limited antioxidant effectiveness have restrained the widespread utilization of nanomedicines in AS treatment. This study aimed to synthesize a specialized peptide-modified liposome capable of encapsulating two antioxidant enzymes, intending to enhance targeted antioxidant therapy for AS. METHODS: The film dispersion method was employed for liposome preparation. Fluorescence quantification was conducted to assess the drug encapsulation rate. Characterization of liposome particle size was performed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Laser confocal microscopy and flow cytometry were utilized to analyze liposome cell uptake and target foam cells. Antioxidant analysis was conducted using 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining, while pro-lipid efflux analysis utilized Oil Red O (ORO) staining. Safety evaluation was performed using Hematoxylin and Eosin (H&E) staining. The level of inflammatory factors was determined through enzyme-linked immunosorbent assay (ELISA). The degree of lipid oxidation at the cellular level was assessed using the malonaldehyde (MDA) assay. In vivo targeting analysis was conducted using small animal live imaging. RESULTS: Our in vitro and in vivo findings substantiated that the modification of Lyp-1 led to increased delivery of antioxidant enzymes into foam cells (p < 0.05), the primary pathological cells within AS plaques. Upon accumulation in foam cells, liposomes loaded with superoxide dismutase (SOD) and catalase (CAT) (LyP-lip@SOD/CAT) effectively mitigated excess ROS and shielded macrophages from ROS-induced damage (p < 0.01). Furthermore, the reduction in ROS levels notably hindered the endocytosis of oxidized low-density lipoprotein (Ox-LDL) by activated macrophages, subsequently alleviating lipid accumulation at atherosclerotic lesion sites, evident from both in vitro and in vivo ORO staining results (p < 0.01). LyP-lip@SOD/CAT significantly curbed the secretion of inflammatory factors at the plaque site (p < 0.001). Additionally, LyP-lip@SOD/CAT demonstrated commendable biological safety. CONCLUSIONS: In this study, we effectively synthesized LyP-lip@SOD/CAT and established its efficacy as a straightforward and promising nano-agent for antioxidant therapy targeting atherosclerosis.

M2 Macrophage-Derived Exosomes Inhibit Apoptosis of HUVEC Cell through Regulating miR-221-3p Expression.

Atherosclerosis (AS) is associated with high morbidity and mortality rates and currently has no effective treatment. This study was aimed at investigating the role of macrophage exosomes in the inflammation and apoptosis after HUVEC injury. We established the HUVEC injury model using 100 mg/L oxidized low-density lipoprotein (ox-LDL) or 50 ng/mL tumor necrosis factor-α (TNF-α). Cell proliferation was assessed using cell counting kit-8 (CCK8) assays, and the expression of miR-221, TNF-α, and IL-6, IL-10, and IL-1ß was detected using quantitative real-time PCR (qRT-PCR). The apoptotic rate was analyzed by the TUNEL method, and the expressions of apoptosis-related proteins Bcl2, Caspase-3, and c-myc were detected by western blotting. Finally, miR-221-3p mimics and miR-221-3p inhibitors were constructed by liposome transfection to determine the mechanism of action of macrophage exosomes on HUVEC injury. The expression levels of IL-6, IL-1ß, and TNF-α in the injury groups were higher than those in the normal group, but the expression of IL-10 in the injury groups was lower than that in the normal group. Meanwhile, the apoptotic rate of the HUVEC cell injury group was higher than that of the normal group. In contrast, the expression levels of IL-6, IL-1ß, and TNF-α were lower in the M2 macrophage exosome (M2-Exo) group, but the expression of IL-10 was higher compared with the control group. The apoptosis rate was reduced in the M2-Exo group, and the expression of the proapoptotic gene Caspase-3 was reduced, while the expression of the antiapoptotic gene Bcl2 was increased. Liposome transfection of miR-221-3p mimics was able to enhance the effect of M2 macrophage exosomes. Thus, M2-Exo promotes HUVEC cell proliferation and inhibits HUVEC cell inflammation and apoptosis. miR-221-3p overexpression attenuates HUVEC cell injury-induced inflammatory response and apoptosis, while miR-221-3p gene inhibition enhances this inflammatory response and apoptosis.

Phthalate promotes atherosclerosis through interacting with long-non coding RNA and induces macrophage foam cell formation and vascular smooth muscle damage.

BACKGROUND: Phthalates are commonly used in variety of plastic products. Previously it has been revealed that di (2-ethylhexyl) phthalate (DEHP), as the most common member of the class of phthalates, may disturb cholesterol homeostasis and deregulate the inflammatory response, and leading to accelerate the atherosclerosis process. In this regard, the aim of the current study is to explore the underlying mechanism of DEHP-induced atherosclerosis through the increasing of foam cell formation and Vascular Smooth Muscle Cells (VSMCs) damage via the interaction of long-non coding RNA (GAS5) and miR-145-5p. METHODS: ApoE-/- mice were used to evaluate the in vivo study. RAW264.7 and VSMCs were used to evaluate the effect of DEHP on formation of foam cell, cell proliferation, and cell damage in vitro. Animals were treated with DEHP (5% w/w of food) orally and cells were treated with medium containing of 100 µM DEHP; qRT-PCR, Western blotting, flowcytometry, IHC, oil red O, BODIPY, and autophagic vacuoles assay were used to evaluate the effect of DEHP on formation of atherosclerosis. RESULTS: DEHP significantly accelerated the formation of atherosclerosis in mice and alter the lipid profile in mice. In addition, after treating VSMCs with DEHP, GAS5 was significantly up-regulated and miR-145-5p was down-regulated. In VSMCs treated with DEHP, we observed that GAS5 could be used as the competing endogenous RNA (ceRNA) of miR-145-5p to regulate the proliferation and apoptosis of VSMCs; and the expression of GAS5 was correlated with the expression of miR-145-5p. DEHP increased the ox-LDL uptake by macrophage and increasing the formation of foam cells. Besides, GAS5 knocking down reversed the effect of DEHP on foam cell formation and ox-LDL uptake. CONCLUSION: DEHP could accelerate the atherosclerosis process through increasing VSMCs damage and formation of macrophage foam cell by increasing lipid uptake though down regulating lncRNA GAS5 and altering in regulation of miR-145-5p.

Oxidized low-density lipoprotein-induced periodontal inflammation is associated with the up-regulation of cyclooxygenase-2 and microsomal prostaglandin synthase 1 in human gingival epithelial cells.

Periodontitis is characterized by chronic gingival tissue inflammation, and inflammatory mediators such as IL-8 and prostaglandin E(2) (PGE(2)) are associated with disease progression. Previously we showed that oxidatively modified low-density lipoprotein (oxLDL) was present in gingival crevicular fluid. In this study, the role of oxLDL in the gingival epithelial cell inflammatory response was further investigated using Ca9-22 cells and primary human oral keratinocytes (HOK). Treatment of Ca9-22 cells and HOK with oxLDL induced an up-regulation of IL-8 and the PGE(2)-producing enzymes, cyclooxygenase-2 and microsomal PGE(2) synthase-1. These responses induced by oxLDL were significantly suppressed by a nuclear factor-kappa B (NF-κB) inhibitor. However, unlike the result in macrophages, oxLDL did not lead to an increase in CD36 expression in these two cells. These results suggest that oxLDL elicits gingival epithelial cell inflammatory responses through an activation of the NF-κB pathway. These data suggest a mechanistic link between periodontal disease and lipid metabolism-related disorders, including atherosclerosis.

Porphyromonas gingivalis lipopolysaccharide alters atherosclerotic-related gene expression in oxidized low-density-lipoprotein-induced macrophages and foam cells.

BACKGROUND AND OBJECTIVE: The molecular mechanism linking atherosclerosis formation and periodontal pathogens is not clear, although a positive correlation between periodontal infections and cardiovascular diseases has been reported. The aim of this study was to determine whether stimulation with Porphyromonas gingivalis lipopolysaccharide (LPS) affected the expression of atherosclerosis-related genes, during and after the formation of foam cells. MATERIAL AND METHODS: Macrophages from human THP-1 monocytes were treated with oxidized low-density lipoprotein (oxLDL) to induce the formation of foam cells. P. gingivalis LPS was added to cultures of either oxLDL-induced macrophages or foam cells. The expression of atherosclerosis-related genes was assayed by quantitative real-time PCR, and the production of granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1, interleukin (IL)-1ß, IL-10 and IL-12 proteins was determined using ELISA. Nuclear translocation of nuclear factor-kappaB (NF-κB) P(65) was detected by immunocytochemistry, and western blotting was used to evaluate inhibitory kappa B-α (IκΒ-α) degradation to confirm activation of the NF-κB pathway. RESULTS: P. gingivalis LPS stimulated atherosclerosis-related gene expression in foam cells and increased the oxLDL-induced expression of chemokines, adhesion molecules, growth factors, apoptotic genes and nuclear receptors in macrophages. Transcription of the proinflammatory cytokines IL1ß and IL12 was elevated in response to LPS in both macrophages and foam cells, whereas transcription of the anti-inflammatory cytokine, IL10, was not affected. Increased activation of the NF-κB pathway was also observed in macrophages costimulated with LPS + oxLDL. CONCLUSION: P. gingivalis LPS appears to be an important factor in the development of atherosclerosis by stimulation of atherosclerosis-related gene expression in both macrophages and foam cells via activation of the NF-κB pathway.

Modulation of apoprotein E secretion in response to receptor-mediated endocytosis in resident and inflammatory macrophages.

We have determined the effect of various endocytic ligands on the secretion of ApoE by macrophages. ApoE was a major secreted protein of resident macrophages, but BCG-activated macrophages secreted little ApoE and periodate-elicited macrophages secreted intermediate amounts of ApoE. Resident, periodate-elicited, and BCG-activated mouse peritoneal macrophages were incubated with AcLDL, EIgG, EIgMC, dextran sulfate, latex, or zymosan, and the resulting protein secretion patterns were analyzed by [35S]methionine labeling and SDS-polyacrylamide gel electrophoresis. AcLDL increased total [35S]methionine incorporation into secreted proteins. Although AcLDL increased the secretion of ApoE by resident macrophages less than or equal to fivefold in a dose-dependent manner, with maximal stimulation at 4.8 micrograms/ml, it decreased the secretion of ApoE by periodate-elicited macrophages to almost nothing and did not affect the low rate of secretion of ApoE by BCG-activated macrophages. However, EIgG, which increases cellular cholesterol content of macrophages as AcLDL does, did not increase ApoE secretion, and dextran sulfate, which is recognized by the same receptor as AcLDL, also did not increase ApoE secretion. The binding and uptake of EIgG, dextran sulfate, zymosan, latex, and EIgMC all decreased the secretion of ApoE. These endocytic ligands also altered the pattern of secreted and cellular proteins other than ApoE. The pattern of response was ligand-specific. However, increased secretion of polypeptides of Mr 62,000 and 68,000 was common to many stimuli. We conclude that receptor-mediated endocytosis modulates the secretion of ApoE and other proteins pleiotypically in resident, inflammatory, and activated macrophages.

Oxidized low-density lipoprotein increases interleukin-8 production in human gingival epithelial cell line Ca9-22.

BACKGROUND AND OBJECTIVE: Recent epidemiological studies have shown a correlation between periodontitis and hyperlipidemia. We have found high levels of oxidized low-density lipoprotein (OxLDL) in the gingival crevicular fluid of dental patients. In the present study, we tried to examine the possible role of OxLDL in periodontal inflammation in vitro. MATERIAL AND METHODS: Cells of the human gingival epithelial cell line Ca9-22 were cultured in media containing OxLDL, and the amounts of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2)) produced were measured using ELISAs. RESULTS: Production of IL-8 by Ca9-22 cells was significantly increased when the cells were treated with OxLDL, but not with native LDL or acetylated LDL. Production of PGE(2) by Ca9-22 cells was enhanced by co-incubation with OxLDL and interleukin-1 beta (IL-1 beta). Scavenger receptor inhibitors, fucoidan and dextran sulfate, inhibited the OxLDL-induced IL-8 and PGE(2) production in the presence of IL-1 beta. The p(38) MAPK inhibitors SB203580 and SB202190 and the ERK inhibitor PD98059 inhibited the OxLDL-induced IL-8 production. Among oxidized lipids and chemically modified LDL, 7-ketocholesterol enhanced IL-8 production. CONCLUSION: This is the first report to show that OxLDL enhances IL-8 production in epithelial cells.

Coatings of low-density lipoprotein and synthetic glycoconjugates as substrata for hepatocytes.

Asialoglycoprotein (ASGP) receptors expressed on rat hepatocytes interact with glycoproteins containing galactose or N-acetylgalactosamine residues at the nonreducing termini of oligosaccharide chains to mediate endocytosis, and cholesterol transport protein with apolipoprotein B (LDL, low-density lipoprotein) in plasma interacts with LDL receptors and heparinoids in the extracellular matrix. We developed novel techniques to prepare galactose- and LDL-immobilized culture plates, using galactose-tagged polystyrene (galactose-carrying polystyrene [GalCPS]: N-p-vinylbenzyl-O-beta-D-galactopyranosyl-[1-->4]-D-gluconamide) and poly(2-acrylamide-2-methyl-1-propanesulfonate) (PAPS), respectively. Hepatocytes adhered well to plates coated with either GalCPS or LDL, and therefore the GalCPS- and LDL-coated plates were examined as specific substrata for culturing hepatocytes. These cultures promoted the formation of three-dimensional, multicellular aggregates with regulation of excess proliferation of non-parenchymal cells. Furthermore, the LDL coating resulted in higher albumin synthesis and an identical level of lactate dehydrogenase (LDH) compared with cells cultured on collagen- and GalCPS-coated plates. Thus, the two culture systems described here, and especially the LDL-coated plates, have potential for the development of a hybrid artificial liver.

Gingival mesenchymal stem cells attenuate pro-inflammatory macrophages stimulated with oxidized low-density lipoprotein and modulate lipid metabolism.

OBJECTIVE: To examine the effects of gingival mesenchymal stem cells (GMSCs) on inflammatory macrophages upon oxidized low-density lipoprotein (ox-LDL) stimulation and evaluate therapeutic potential of GMSCs on mouse model of periodontitis associated with hyperlipidemia. METHODS: in vitro, GMSCs were co-cultured with macrophages for 48 h in the absence or presence of M1 polarizing conditions and oxidized low-density lipoprotein in the transwell system. The supernatants were collected for ELISA. M1 and M2 markers of macrophages were analyzed by flow cytometry and PCR, and lipid accumulation was assessed by oil red O staining. in vivo, eighteen mice were divided into three groups (n = 6): Group A (periodontally healthy mice as control), Group B (periodontitis mice with hyperlipidemia), Group C (periodontitis mice with hyperlipidemia with the transplantation of GMSCs). The serum levels of cholesterol and inflammatory factors were measured by automatic analyzer. Bone regeneration was evaluated by Masson staining. RESULTS: When co-cultured with GMSCs, the M1 markers of Tumor Necrosis Factor (TNF) -α, Interleukin (IL) -6, Interleukin (IL) -1ß, CD86, and Human Leukocyte Antigen (HLA) -DR were significantly reduced. In contrast, M2 markers such as Interleukin(IL) -10 and CD206 were moderately increased. Similar results were obtained in the cell culture supernatants. In animal experiment, GMSCs suppressed the expression of sterol regulatory element binding transcription factor 1c (SREBP-1c) and elevated the levels of peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator activator receptor- coactivator 1(PGC-1α) in the liver, attenuated cholesterol dysfunction via the downregulation of low-density lipoprotein (LDL) and total cholesterol (TC), and the upregulation of high-density lipoprotein (HDL), and decreased the levels of TNF-α and IL-6. Moreover, GMSC treatment improved bone regeneration. CONCLUSION: GMSCs inhibit the activation of M1 macrophages, regulate lipid metabolism and reduce inflammatory response, and promote bone regeneration in mouse model of periodontitis associated with hyperlipidemia.

Porphyromonas gingivalis-induced inflammatory responses in THP1 cells are altered by native and modified low-density lipoproteins in a strain-dependent manner.

Strong epidemiological evidence supports an association between cardiovascular and periodontal disease and furthermore, the periodontopathogen Porphyromonas gingivalis has been identified in blood and from atheromatous plaques. Blood exposed to P. gingivalis shows an increased protein modification of low-density lipoprotein (LDL). In this study, we investigate the inflammatory responses of THP1 cells incubated with P. gingivalis and the effects of native or modified LDL on these responses. Reactive oxygen species (ROS) and IL-1ß were observed in THP1 cells following infection with P. gingivalis ATCC33277 and W50. Caspase 1 activity was quantified in THP1 cells and correlated with IL-1ß accumulation. Oxidized LDL (oxLDL) induced IL-1ß release and CD36 expression on THP1 cells. Modified LDL co-stimulated with ATCC33277 exhibited regulatory effects on caspase 1 activity, IL-1ß release and CD36 expression in THP1 cells, whereas W50 induced more modest responses in THP1 cells. In summary, we show that P. gingivalis is capable of inducing pro-inflammatory responses in THP1 cells, and native and modified LDL could alter these responses in a dose- and strain-dependent manner. Strain-dependent differences in THP1 cell responses could be due to the effect of P. gingivalis proteases, presence or absence of capsule and proteolytic transformation of native and modified LDL.

Oxidized Lipoprotein as a Major Vessel Cell Proliferator in Oxidized Human Serum.

Oxidative stress is correlated with the incidence of several diseases such as atherosclerosis and cancer, and oxidized biomolecules have been determined as biomarkers of oxidative stress; however, the detailed molecular relationship between generated oxidation products and the promotion of diseases has not been fully elucidated. In the present study, to clarify the role of serum oxidation products in vessel cell proliferation, which is related to the incidence of atherosclerosis and cancer, the major vessel cell proliferator in oxidized human serum was investigated. Oxidized human serum was prepared by free radical exposure, separated using gel chromatography, and then each fraction was added to several kinds of vessel cells including endothelial cells and smooth muscle cells. It was found that a high molecular weight fraction in oxidized human serum specifically induced vessel cell proliferation. Oxidized lipids were contained in this high molecular weight fraction, while cell proliferation activity was not observed in oxidized lipoprotein-deficient serum. Oxidized low-density lipoproteins induced vessel cell proliferation in a concentration-dependent manner. Taken together, these results indicate that oxidized lipoproteins containing lipid oxidation products function as a major vessel cell proliferator in oxidized human serum. These findings strongly indicate the relevance of determination of oxidized lipoproteins and lipid oxidation products in the diagnosis of vessel cell proliferation-related diseases such as atherosclerosis and cancer.

Porphyromonas gingivalis Differentially Modulates Cell Death Profile in Ox-LDL and TNF-α Pre-Treated Endothelial Cells.

OBJECTIVE: Clinical studies demonstrated a potential link between atherosclerosis and periodontitis. Porphyromonas gingivalis (Pg), one of the main periodontal pathogen, has been associated to atheromatous plaque worsening. However, synergism between infection and other endothelial stressors such as oxidized-LDL or TNF-α especially on endothelial cell (EC) death has not been investigated. This study aims to assess the role of Pg on EC death in an inflammatory context and to determine potential molecular pathways involved. METHODS: Human umbilical vein ECs (HUVECs) were infected with Pg (MOI 100) or stimulated by its lipopolysaccharide (Pg-LPS) (1µg/ml) for 24 to 48 hours. Cell viability was measured with AlamarBlue test, type of cell death induced was assessed using Annexin V/propidium iodide staining. mRNA expression regarding caspase-1, -3, -9, Bcl-2, Bax-1 and Apaf-1 has been evaluated with RT-qPCR. Caspases enzymatic activity and concentration of APAF-1 protein were evaluated to confirm mRNA results. RESULTS: Pg infection and Pg-LPS stimulation induced EC death. A cumulative effect has been observed in Ox-LDL pre-treated ECs infected or stimulated. This effect was not observed in TNF-α pre-treated cells. Pg infection promotes EC necrosis, however, in infected Ox-LDL pre-treated ECs, apoptosis was promoted. This effect was not observed in TNF-α pre-treated cells highlighting specificity of molecular pathways activated. Regarding mRNA expression, Pg increased expression of pro-apoptotic genes including caspases-1,-3,-9, Bax-1 and decreased expression of anti-apoptotic Bcl-2. In Ox-LDL pre-treated ECs, Pg increased significantly the expression of Apaf-1. These results were confirmed at the protein level. CONCLUSION: This study contributes to demonstrate that Pg and its Pg-LPS could exacerbate Ox-LDL and TNF-α induced endothelial injury through increase of EC death. Interestingly, molecular pathways are differentially modulated by the infection in function of the pre-stimulation.

In vitro regulation of cholesterol metabolism by low density lipoproteins in skin fibroblasts from hypo-and hyperresponding squirrel monkeys.

When squirrel monkeys (Saimiri sciureus) are fed diets containing cholesterol, some individuals (hyperresponders) become hypercholesterolemic, while others (hyporesponders) are able to maintain nearly normal plasma cholesterol concentrations. Skin fibroblasts were grown from three hyperresponder and threehyporesponder squirrel monkeys, previously characterized on the basis of their plasma cholesterol response to two cholesterol-containing diets and the pheno-type of their parents. The rates of cholesterol synthesis and esterification were determined in the cultured fibroblasts incubated with low density lipoproteins isolated from normocholesterolemic squirrel monkeys or hypercholesterolemic rhesus monkeys. Both lipoprotein preparations influenced the metabolic parameters measured in a similar manner in cells from both hypo- and hyperresponder animals. Exposure of skin fibroblasts to low density lipoproteins resultd in a stimulation of cholesterol esterification and a suppression of cholesterol synthesis in cells from both hypo- and hyperresponder animals. When incubated with increasing concentrations of low density lipoprotein cholesterol, up to 50 microgram/ml, fibroblasts from both hypo-and hyperresponding animals responded with a similar maximum percentage suppression of sterol synthesis. Thus, hyperresponsiveness to dietary cholesterol in squirrel monkeys, although a heritable characteristic, is not associated with an inability of low density lipoprotein to suppress cholesterol synthesis or stimulate cholesterol esterification as occurs in familial hypercholesterolemia in man.

Receptor-mediated endocytosis of poly(acrylic acid)-conjugated liposomes by macrophages.

The uptake characteristics of negatively-charged liposomes made by conjugation of poly(acrylic acid) (PAA) were studied with respect to cultured RAW macrophages. The PAA-conjugated liposomes were internalized and digested in an acidic compartment at a much faster rate than the unmodified phosphatidylcholine (PC) liposomes. After incubation for 18 h, an over 5-fold increase in the uptake of PC liposomes was obtained by PAA conjugation. Subsequently, part of the aqueous phase of the internalized liposomes was exocytosed. Recognition of PAA by the macrophages seems to be responsible for the enhanced uptake of PAA-conjugated liposomes. Cross-competition experiments showed that PAA-conjugated liposomes inhibited the uptake of acetylated-low density lipoprotein (acetyl-LDL) by the macrophages and vice versa. The uptake of PAA-conjugated liposomes was also inhibited by dextran sulfate and maleylated-bovine serum albumin (maleyl-BSA), which are also known to bind to scavenger receptors. Poly(C) and BSA, which are not ligands for the scavenger receptor, competed poorly with the uptake of PAA-conjugated liposomes. Enhanced uptake of PAA-conjugated liposomes by CHO cells with low scavenger receptor expression was not observed. Unexpectedly, LDL, which is not a ligand for scavenger receptor, also partially inhibited the uptake of PAA-conjugated liposomes. The interaction of PAA-conjugated liposomes with macrophages is complex, and the endocytosis of PAA-conjugated liposomes most likely involves multiple receptors and/or pathways. The data obtained suggest that the high affinity binding of PAA-conjugated liposomes to macrophages may be due to recognition of the negative charges of PAA by cell surface receptors, including the scavenger receptor.

Potentiation of beta-amyloid polymerisation by low-density lipoprotein enhances the peptide's vasoactivity.

Alzheimer's disease (AD) is characterised by the accumulation of insoluble beta-amyloid (A beta) fibrils in the brain. Factors that promote A beta fibrillogenesis may influence the pathogenesis of AD and represent targets for therapeutic intervention. Some A beta deposited in AD may originate in the circulation and plasma factors could promote A beta deposition, particularly in the cerebrovasculature. We investigated the effects of plasma low-density lipoprotein (LDL), in both its native and oxidised forms, on A beta(1-40) fibrillogenesis and vasoactivity. LDL enhanced A beta fibrillogenesis in a process dependent on LDL concentration and the oxidative state of the lipoprotein, as indicated by measurements of thiobarbituric acid reactive substances (TBARS) and conjugated dienes. LDL's actions were inhibited by the iA beta 5 peptide, suggesting that LDL-induced A beta polymerisation involved beta-pleated sheet formation. Potentiated A beta polymerisation was reflected by enhanced A beta-mediated vascular responses. Human endothelial cells exposed to fibrillar A beta generated with LDL, especially oxidised LDL, exhibited decreased 20S proteasome activity. Rat aortic ring constriction induced by noradrenaline was enhanced by A beta fibrils generated with LDL, with oxidised LDL producing the more marked effects. Should plasma lipoproteins prove to play a role in cerebral A beta deposition their modification with statins or antioxidants may offer therapeutic benefit.

Low-density lipoprotein upregulates low-density lipoprotein receptor-related protein expression in vascular smooth muscle cells: possible involvement of sterol regulatory element binding protein-2-dependent mechanism.

BACKGROUND: Low-density lipoprotein (LDL) receptor-related protein (LRP) is highly expressed in vascular smooth muscle cells (VSMCs) of both normal and atherosclerotic lesions. However, little is known about LRP regulation in the vascular wall. METHODS AND RESULTS: We analyzed the regulation of LRP expression in vitro in human VSMCs cultured with native LDL (nLDL) or aggregated LDL (agLDL) by semiquantitative reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, and Western blot and in vivo during diet-induced hypercholesterolemia by in situ hybridization. LRP expression in human VSMCs is increased by nLDL and agLDL in a time- and dose-dependent manner. Maximal induction of LRP mRNA expression was observed after 24 hours of exposure to LDL. However, agLDL induced higher LRP mRNA expression (3.0-fold) than nLDL (1.76-fold). LRP mRNA upregulation was associated with an increase on LRP protein expression with the greatest induction by agLDL. VSMC-LRP upregulation induced by nLDL or agLDL was reduced by an inhibitor of sterol regulatory element binding protein (SREBP) catabolism (N-acetyl-leucyl-leucyl-norleucinal). In situ hybridization analysis indicates that there is a higher VSMC-LRP expression in hypercholesterolemic than in normocholesterolemic pig aortas. CONCLUSIONS: These results indicate that LRP expression in VSMCs is upregulated by intravascular and systemic LDL.

Endocytosis of sulfatides by macrophages: relationship to the cellular uptake of phosphatidylserine.

These studies initially examined the effect of sulfatides on the endocytosis of phosphatidylserine (PS) liposomes in J774 macrophages employing liposomes composed entirely of PS and the nonexchangeable radiolabel [3H]cholesteryl hexadecyl ether. Bovine brain sulfatides significantly inhibited the uptake of PS liposomes by macrophages to a level of approximately 15% of control values. To examine whether macrophages were also capable of recognizing and internalizing sulfatides, sulfatide-containing liposomes were prepared using phosphatidylcholine (PC), cholesterol, and sulfatides (6:2:4 molar ratio) incorporating the same radiolabel. The sulfatide-containing liposomes were found to be avidly endocytosed by macrophages. Uptake of the sulfatide-containing liposomes by macrophages was significantly greater than the uptake of liposomes made without sulfatides. When the macrophages were incubated with the anionic compounds dextran sulfate and fucoidin, both the binding of the liposomes to the macrophages at 4 degrees C and the internalization of the liposomes at 37 degrees C were inhibited to approximately 10% of control values. The negatively charged phospholipids phosphatidylglycerol and cardiolipin significantly inhibited the uptake of sulfatide-containing liposomes, and PS was not effective in reducing the cellular uptake of these liposomes. Both oxidized low-density lipoprotein (LDL) and acetylated LDL reduced the uptake of the sulfatide-containing liposomes; the uptake observed in the presence of acetylated LDL and oxidized LDL was approximately 70% and 40%, respectively, of control values. These findings demonstrate that macrophages can efficiently endocytose both sulfatides and negatively charged phospholipids to remove them from the circulation.

CD36/SR-B2-TLR2 Dependent Pathways Enhance Porphyromonas gingivalis Mediated Atherosclerosis in the Ldlr KO Mouse Model.

There is strong epidemiological association between periodontal disease and cardiovascular disease but underlying mechanisms remain ill-defined. Because the human periodontal disease pathogen, Porphyromonas gingivalis (Pg), interacts with innate immune receptors Toll-like Receptor (TLR) 2 and CD36/scavenger receptor-B2 (SR-B2), we studied how CD36/SR-B2 and TLR pathways promote Pg-mediated atherosclerosis. Western diet fed low density lipoprotein receptor knockout (Ldlr°) mice infected orally with Pg had a significant increase in lesion burden compared with uninfected controls.This increase was entirely CD36/SR-B2-dependent, as there was no significant change in lesion burden between infected and uninfected Cd36o/Ldlro mice [corrected]. Western diet feeding promoted enhanced CD36/SR-B2-dependent IL1ß generation and foam cell formation as a result of Pg lipopolysaccharide (PgLPS) exposure. CD36/SR-B2 and TLR2 were necessary for inflammasome activation and optimal IL1ß generation, but also resulted in LPS induced lethality (pyroptosis). Modified forms of LDL inhibited Pg-mediated IL1ß generation in a CD36/SR-B2-dependent manner and prevented pyroptosis, but promoted foam cell formation. Our data show that Pg infection in the oral cavity can lead to significant TLR2-CD36/SR-B2 dependent IL1ß release. In the vessel wall, macrophages encountering systemic release of IL1ß, PgLPS and modified LDL have increased lipid uptake, foam cell formation, and release of IL1ß, but because pyroptosis is inhibited, this enables macrophage survival and promotes increased plaque development. These studies may explain increased lesion burden as a result of periodontal disease, and suggest strategies for development of therapeutics.

Reconstituted high density lipoprotein reduces the capacity of oxidatively modified low density lipoprotein to accumulate cholesteryl esters in mouse peritoneal macrophages.

Oxidized low density lipoprotein (ox-LDL) was incubated with discoidal complexes of apolipoprotein A-I (apo A-I) and dimyristoylphosphatidylcholine (DMPC) (DMPC/apo A-I) in a cell-free system and re-isolated on Sephacryl S-400 gel filtration chromatography. Analyses of re-isolated ox-LDL showed that apo A-I was transferred from DMPC/apo A-I to ox-LDL, which accounted for 10% of the total protein of ox-LDL. Re-isolated ox-LDL also showed a 2.2-fold increase in phospholipid and a 14% decrease in cholesterol content on an apo B basis. The electrophoretic mobility of re-isolated ox-LDL was markedly reduced almost to that of native LDL. Moreover, the amounts of re-isolated ox-LDL to be degraded by mouse peritoneal macrophages as well as the capacity of re-isolated ox-LDL to accumulate cholesteryl esters (CE) in these cells were markedly reduced (60% and 80% reduction, respectively), suggesting that the ligand activity of ox-LDL for the scavenger receptor was significantly reduced upon treatment with DMPC/apo A-I. Parallel incubation of ox-LDL with free apo A-I led to a similar incorporation of apo A-I into ox-LDL. However, it had no effects on the ligand activity of ox-LDL. Thus, it is likely that the reduction in the ligand activity of ox-LDL by DMPC/apo A-I is explained by the change in the lipid moiety (mainly phospholipid) of ox-LDL. Since discoidal high density lipoprotein (HDL) is known to occur in vivo, this phenomenon might explain one of the anti-atherogenic functions of HDL.

Porphyromonas gingivalis induces its uptake by human macrophages and promotes foam cell formation in vitro.

Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans, which has been linked to an increased risk for atherosclerosis-related events. In this study, we examined the effect of P. gingivalis infection on human macrophages with respect to foam cell formation, the hallmark of early atherogenesis, and the potential of P. gingivalis to induce its uptake by these cells. Human monocyte-derived macrophages were incubated with low density lipoprotein and infected with P. gingivalis FDC381 or its fimbriae deficient mutant, DPG3. Consistent with a role for fimbriae in this process, strain 381 significantly increased foam cell formation as compared to DPG3. Recovery of viable P. gingivalis in antibiotic protection experiments was significantly higher for strain 381 than for DPG3. By transmission electron microscopy, the wild-type strain was shown to adhere to and enter THP-1 cells. These results suggest that properties of P. gingivalis which render it capable of adhering to/invading other cell types may also be operative in macrophages and play an important role in its atherogenic potential.