PubMed Health⌕ Search

Biomedical subjects

J A Berliner

Publications and source records attributed to J A Berliner.

At least 19 recordsLinked to original sources
PubMed2001

Rantes production during development of cardiac allograft vasculopathy.

BACKGROUND: RANTES (regulated on activation, normal T cell expressed and secreted) production has been shown to correlate with mononuclear cell recruitment and precede intimal thickening in cardiac allograft vasculopathy (CAV). However, the cells that produce RANTES in CAV are undefined. Therefore, in an MHC II-mismatched murine model of CAV, we sought to (1) define the cellular sources of RANTES and (2) determine the role of CD4+ lymphocytes in RANTES production during CAV development. METHODS: B6.CH-2bm12 strain donor hearts were transplanted heterotopically into wild-type (WT) or CD4 knockout (CD4KO) C57BL/6 mice (MHC II mismatch). No immunosuppression was used. Recipients were sacrificed at 7, 14, and 24 days. Intragraft RANTES gene expression and protein levels were determined with ribonuclease protection assay and ELISA, respectively. At days 7 and 24, RANTES production by graft-infiltrating cells was defined with intracellular RANTES staining and multicolor FACS analysis. Intimal thickening was quantitated morphometrically. In murine hearts and in six explanted human hearts with advanced CAV, RANTES was also localized immunohistochemically. RESULTS: NK, NKT, and gammadelta+ cells, in addition to CD4+, CD8+ lymphocytes, and CD11b+ macrophages, produced RANTES in early and late stages of CAV. RANTES-producing NK, NKT, and gammadelta+ cells tripled in number during CAV development; by day 24, NK and gammadelta+ cells each outnumbered CD4+ lymphocytes and CD11b+ macrophages. The presence of CD4+ lymphocytes was required for sustained RANTES production in allografts, which correlated with mononuclear cell recruitment and preceded intimal thickening. In murine and explanted human hearts with advanced CAV, RANTES immunolocalized with graft-infiltrating mononuclear cells and vessel wall cells. CONCLUSIONS: We present evidence that other cell types in addition to CD4+, CD8+ T lymphocytes, and CD11b+ macrophages contribute significantly to RANTES production in CAV. In this MHC II-mismatched murine model of CAV, sustained RANTES production requires CD4+ lymphocytes, correlates with mononuclear cell recruitment, and precedes intimal thickening. In experimental and human CAV, vessel wall cells may also produce RANTES. Interventions aimed at inhibiting RANTES production in CAV may need to target several types of cells, and neutralization of RANTES bioactivity may reduce mononuclear cell recruitment and CAV development.

Animals↗
PubMed2001

Adenoviral delivery of a leukocyte-type 12 lipoxygenase ribozyme inhibits effects of glucose and platelet-derived growth factor in vascular endothelial and smooth muscle cells.

The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2001

HDL and the inflammatory response induced by LDL-derived oxidized phospholipids.

Oxidation of low density lipoprotein (LDL) phospholipids containing arachidonic acid at the sn-2 position occurs when a critical concentration of "seeding molecules" derived from the lipoxygenase pathway is reached in LDL. When this critical concentration is reached, the nonenzymatic oxidation of LDL phospholipids produces a series of biologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. Normal high density lipoprotein (HDL) contains at least 4 enzymes as well as apolipoproteins that can prevent the formation of the LDL-derived oxidized phospholipids or inactivate them after they are formed. In the sense that normal HDL can prevent the formation of or inactivate these inflammatory LDL-derived oxidized phospholipids, normal HDL is anti-inflammatory. HDL from mice that are genetically predisposed to diet-induced atherosclerosis became proinflammatory when the mice are fed an atherogenic diet, injected with LDL-derived oxidized phospholipids, or infected with influenza A virus. Mice that were genetically engineered to be hyperlipidemic on a chow diet and patients with coronary atherosclerosis, despite normal lipid levels, also had proinflammatory HDL. It is proposed that LDL-derived oxidized phospholipids and HDL may be part of a system of nonspecific innate immunity and that the detection of proinflammatory HDL may be a useful marker of susceptibility to atherosclerosis.

Animals↗
PubMed2001

Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) upregulates a spectrum of inflammatory cytokines and adhesion molecules different from those induced by classic inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide. Interestingly, Ox-PAPC also induces the expression of a set of proteins similar to those induced by TNF-alpha or lipopolysaccharide, which include the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8. To elucidate the molecular mechanisms of Ox-PAPC-induced gene expression and to determine whether Ox-PAPC and other inflammatory mediators such as TNF-alpha utilize common signaling pathways, we examined the transcriptional regulation of IL-8 by Ox-PAPC and TNF-alpha in human aortic endothelial cells. Both Ox-PAPC and TNF-alpha induced the expression of IL-8 mRNA in a dose-dependent fashion; however, the kinetics of IL-8 mRNA accumulation between the 2 ligands differed. Ox-PAPC-induced IL-8 mRNA was seen as early as 30 minutes, peaked between 4 and 8 hours, and decreased substantially by 24 hours. In contrast, TNF-alpha-induced IL-8 mRNA synthesis was elevated at 30 minutes, peaked at 2 hours, and reached basal/undetectable levels by 6 hours. Actinomycin D experiments suggested that both Ox-PAPC and TNF-alpha regulate the expression of IL-8 at the transcriptional level. Furthermore, the half-life of IL-8 mRNA for both ligands was similar (<30 minutes), suggesting that mRNA stability was not responsible for the differences in the kinetics of IL-8 accumulation between the 2 ligands. Transient transfection studies with reporter constructs containing 1.48 kb of the IL-8 promoter identified an Ox-PAPC-specific response region between -133 and -1481 bp of the IL-8 promoter. In contrast, TNF-alpha activation of the IL-8 promoter was mediated almost entirely through the nuclear factor-kappaB and activation protein-1 response elements present between -70 and -133 bp of the IL-8 promoter. Thus, although Ox-PAPC and TNF-alpha both induced IL-8 synthesis, our data suggest that the 2 ligands utilize different mechanisms in the regulation of IL-8 transcription.

Cells, Cultured↗
PubMed2000

Role for peroxisome proliferator-activated receptor alpha in oxidized phospholipid-induced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells.

The attraction, binding, and entry of monocytes into the vessel wall play an important role in atherogenesis. We have previously shown that minimally oxidized/modified LDL (MM-LDL), a pathogenically relevant lipoprotein, can activate human aortic endothelial cells (HAECs) to produce monocyte chemotactic activators. In the present study, we demonstrate that MM-LDL and oxidation products of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) activate endothelial cells to synthesize monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8). Several lines of evidence suggest that this activation is mediated by the lipid-dependent transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha), the most abundant member of the PPAR family in HAECs. Treatment of transfected CV-1 cells demonstrated activation of the PPARalpha ligand-binding domain by MM-LDL, Ox-PAPC, or its component phospholipids, 1-palmitoyl-2-oxovalaroyl-sn-glycero-phosphocholine and 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine; these lipids also activated a consensus peroxisome proliferator-activated receptor response element (PPRE) in transfected HAECs. Furthermore, activation of PPARalpha with synthetic ligand Wy14,643 stimulates the synthesis of IL-8 and MCP-1 by HAECs. By contrast, troglitazone, a PPARgamma agonist, decreased the levels of IL-8 and MCP-1. Finally, we demonstrate that unlike wild-type endothelial cells, endothelial cells derived from PPARalpha null mice do not produce MCP-1/JE in response to Ox-PAPC and MM-LDL. Together, these data demonstrate a proinflammatory role for PPARalpha in mediation of the activation of endothelial cells to produce monocyte chemotactic activity in response to oxidized phospholipids and lipoproteins.

Animals↗
PubMed2000

Bioactive products of phospholipid oxidation: isolation, identification, measurement and activities.

There is considerable evidence to suggest that oxidation of LDL plays an important role in atherogenesis. Polyunsaturated fatty acids, a major oxidative target, are present as phospholipids in the outer core of the lipoprotein particle. Studies from several laboratories have shown an increase in the levels of phospholipid oxidation products in atherosclerotic lesions and of antibodies to oxidized phospholipids in mice and humans with lesions. Significantly, phospholipid oxidation products have been demonstrated (in vitro) to selectively activate processes in vascular wall cells that may contribute to atherogenesis. This review discusses activities, methods for isolation, identification and measurement of bioactive phospholipids. Past studies suggest that defined and relatively simple current technologies allow identification of bioactive phospholipid oxidation products and measurement of their levels in tissue.

Animals↗
PubMed2000

Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis.

Serum paraoxonase (PON1), present on high density lipoprotein, may inhibit low density lipoprotein (LDL) oxidation and protect against atherosclerosis. We generated combined PON1 knockout (KO)/apolipoprotein E (apoE) KO and apoE KO control mice to compare atherogenesis and lipoprotein oxidation. Early lesions were examined in 3-month-old mice fed a chow diet, and advanced lesions were examined in 6-month-old mice fed a high fat diet. In both cases, the PON1 KO/apoE KO mice exhibited significantly more atherosclerosis (50-71% increase) than controls. We examined LDL oxidation and clearance in vivo by injecting human LDL into the mice and following its turnover. LDL clearance was faster in the double KO mice as compared with controls. There was a greater rate of accumulation of oxidized phospholipid epitopes and a greater accumulation of LDL-immunoglobulin complexes in the double KO mice than in controls. Furthermore, the amounts of three bioactive oxidized phospholipids were elevated in the endogenous intermediate density lipoprotein/LDL of double KO mice as compared with the controls. Finally, the expression of heme oxygenase-1, peroxisome proliferator-activated receptor gamma, and oxidized LDL receptors were elevated in the livers of double KO mice as compared with the controls. These data demonstrate that PON1 deficiency promotes LDL oxidation and atherogenesis in apoE KO mice.

Animals↗
PubMed2000

Determinants of bioactivity of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position.

We previously described 3 bioactive oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) containing oxovaleroyl (POVPC), glutaroyl (PGPC), and epoxyisoprostane (PEIPC) groups at the sn-2 position that were increased in minimally modified/oxidized low density lipoprotein (MM-LDL) and rabbit atherosclerotic lesions. We demonstrated specific and contrasting effects of POVPC and PGPC on leukocyte-endothelial interactions and described an effect of PEIPC on monocyte binding. The major purpose of the present study was to determine the effects of structural changes on the bioactivities of these 3 lipids. We demonstrate herein that the group at the sn-2 position determines the specific bioactivity and that the substitution of stearoyl for palmitoyl at the sn-1 position or ethanolamine for choline at the sn-3 position of the phospholipid did not alter bioactivity. Oxidized PAPC, oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, and oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylethanolamine stimulated monocyte binding and inhibited lipopolysaccharide-induced expression of the neutrophil-binding molecule E-selectin. Furthermore, all oxovaleroyl phospholipids but not the glutaroyl phospholipids induced monocyte binding without an increase in vascular cell adhesion molecule-1 (VCAM-1) expression and inhibited lipopolysaccharide-induced E-selectin expression. In contrast, glutaroyl phospholipids but not oxovaleroyl phospholipids stimulated E-selectin and VCAM-1 expression. We further demonstrate that all parts of the phospholipid molecules are required for these bioactivities. Hydrolysis with phospholipase (PL) A(1), PLA(2), and PLC strongly reduced the bioactivities of POVPC, PGPC, and mixed isomers of PEIPC. PLD had a smaller but still significant effect. The effects of POVPC and PEIPC could be abolished by sodium borohydride treatment, indicating the importance of the reducible groups (carbonyl and epoxide) in these molecules. In summary, these studies identify 6 new bioactive, oxidized phospholipids that are increased in MM-LDL and, where measured, in atherosclerotic lesions. They thus suggest that a family of phospholipid oxidation products containing oxovaleroyl, glutaroyl, and epoxyisoprostane at the sn-2 position play an important role in the regulation of leukocyte-endothelial interactions, bioactivity being in part controlled by several types of phospholipid hydrolases.

Animals↗
PubMed2000

Induction of IG9 monocyte adhesion molecule expression in smooth muscle and endothelial cells after balloon arterial injury in cholesterol-fed rabbits.

The expression of monocyte-specific adhesion molecules and chemokines by cell types within the vessel wall plays an important role in foam cell accumulation during atherosclerotic plaque development. We previously identified IG9, a novel monocyte adhesion protein that is expressed on endothelial cells (ECs) overlying human and rabbit advanced atherosclerotic plaques. The present study was designed to determine the temporal and spatial expression of IG9 and the chemokine, monocyte chemoattractant protein-1 (MCP-1), after balloon injury with (double injury) or without (single injury) prior air desiccation EC injury in the femoral arteries of rabbits fed a high-cholesterol diet. By immunohistochemical analyses, intense reactivity with monoclonal antibodies to IG9 and MCP-1 was detected 24 hours after single injury in medial smooth muscle cells (SMCs) and in SMCs of adventitial microvessels. However, monocyte infiltration of the tunica media was minimal or not detected in these sections. IG9 and MCP-1 antibody reactivity in vessel sections 28 days after single injury and 24 hours, 7 days, and 28 days after double injury was localized to medial and neointimal SMCs, foam cells, and luminal ECs overlying the plaques. Uninjured rabbit (cholesterol or normal diet) vessel sections exhibited minimal IG9 and MCP-1 immunostaining. In vitro studies using human aortic SMCs demonstrated IG9 protein induction after 24 hours of treatment with platelet-derived growth factor-BB and interferon-gamma or epidermal growth factor. IG9 expression was further increased by pretreatment of SMCs with the proatherogenic lipid, minimally oxidized low density lipoprotein. After balloon injury (24 hours), IG9 is induced in vascular SMCs before the detectable accumulation of monocytes within the vessel wall. Thus, the expression of IG9 by SMCs as well as by ECs may be an important factor in the accumulation of foam cells in atherosclerotic plaque development after arterial injury.

Animals↗
PubMed2000

Toxicity of Dutch (E22Q) and Flemish (A21G) mutant amyloid beta proteins to human cerebral microvessel and aortic smooth muscle cells.

BACKGROUND AND PURPOSE: Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid beta protein (Abeta) in cortical and leptomeningeal vessels of patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis, Dutch type. Smooth muscle cells (SMC) from cerebral microvessels (MV) are of particular interest as a site of Abeta-related injury because CAA is much more pronounced in the tunica media of cortical arterioles than meningeal arteries. Patients carrying point mutations at residues 22 (E22Q) and 21 (A21G) of Abeta show severe CAA with various degrees of brain parenchymal Abeta deposition. The purpose of this study was to investigate the effects of 2 mutant E22Q- and A21G-Abeta peptides on MV and aortic SMC. MERHODS: SMC were isolated from human cerebral MV and aorta. Cell morphology, viability, and proliferation as parameters of Abeta toxicity were investigated after 3 days of peptide treatment by trypan blue exclusion and [(3)H]thymidine incorporation. RESULTS: E22Q-Abeta induced significant decreased cellular proliferation and viability, as well as obvious degeneration of both MV and aortic SMC. A21G-Abeta and wild-type Abeta did not cause significant toxicity, as judged by cell morphology, viability, or cell proliferation, on either type of SMC. CONCLUSIONS: E22Q-Abeta induced greater toxicity in all parameters than A21G-Abeta and wild-type Abeta with respect to both MV and aortic SMC. A21G-Abeta did not show a significant toxic effect on MV and aortic SMC. This differential effect may be linked to cell type-specific processing and metabolism of mutant forms of Abeta. Mutations in amyloid precursor protein may lead to CAA by different pathogenetic mechanisms or share an unknown property that distinguishes them from wild-type Abeta.

Amyloid beta-Peptides↗
PubMed2000

Adhesion molecule expression in a rat model of Staphylococcus aureus endophthalmitis.

PURPOSE: To determine whether Staphylococcus aureus and its components induce expression of E-selectin and intercellular adhesion molecule (ICAM)-1 in rat ocular tissues and on human endothelial cells in culture. METHODS: Experimental and control rat eyes were injected with 80 colony-forming units of viable S. aureus and lipopolysaccharide-free sterile saline (NS), respectively. Eyes were enucleated and immediately frozen. E-selectin and ICAM-1 expression were evaluated on frozen sections by using standard immunohistochemical techniques. Using an enzyme-linked immunoassay, in vitro expression of E-selectin and ICAM-1 was evaluated on macrovascular endothelial cells after stimulation with S. aureus and selected purified components. RESULTS: In S. aureus-injected eyes, E-selectin and ICAM-1 expression peaked at six to 24 hours, decreased slightly at 24 and 48 hours, and further declined by 72 hours. However, in NS-injected eyes, peak levels of E-selectin and ICAM-1 were seen at 6 hours, after which expression declined in the areas in which an increase was previously observed. In in vitro assays, peptidoglycan (0.01 microg/ml) induced a fourfold increase in E-selectin (P < 0.0001) and a twofold increase in ICAM-1 (P < 0.002) expression. Ribitol teichoic acid (RTA) (1 microg/ml) induced a twofold increase in E-selectin (P < 0.0001) and a threefold increase in ICAM-1 (P < 0.0001) expression. CONCLUSIONS: Eyes injected with S. aureus demonstrated a more intense and prolonged expression of both E-selectin and ICAM-1 than did eyes injected with NS. In addition, S. aureus components induced the in vitro expression of these adhesion molecules on macrovascular endothelial cells. The relevance of these findings to microvascular endothelial cells is yet to be determined.

Animals↗
PubMed1999

Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils.

We previously have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified low density lipoprotein (MM-LDL), activates endothelial cells to bind monocytes. 1-Palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), which are present in OxPAPC, MM-LDL, and atherosclerotic lesions, were shown to have a major role in the activation of endothelial cells. We now demonstrate that these two highly similar molecules have dramatically different effects on leukocyte endothelial interactions. POVPC is a potent regulator of monocyte-specific endothelial interactions. Treatment of endothelial cells with POVPC increased monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin; no increase in neutrophil binding was observed. In addition, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition was mediated by a protein kinase A-dependent pathway, resulting in down-regulation of NF-kappaB-dependent transcription. In contrast, PGPC induced both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule 1. We present evidence to suggest that the two phospholipids act by different novel receptors present in Xenopus laevis oocytes and that POVPC, but not PGPC, stimulates a cAMP-mediated pathway. At concentrations equal to that present in MM-LDL, the effect of POVPC dominates and inhibits PGPC-induced neutrophil binding and E-selectin expression in endothelial cells. In summary, our data provide evidence that both POVPC and PGPC are important regulators of leukocyte-endothelial interactions and that POVPC may play a dominant role in a number of chronic inflammatory processes where oxidized phospholipids are known to be present.

Aorta↗
PubMed1999

Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.

One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A(2) and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5, 6-epoxyisoprostane E(2))-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.

Arachidonic Acids↗
PubMed1999

Evidence that phospholipid oxidation products and/or platelet-activating factor play an important role in early atherogenesis : in vitro and In vivo inhibition by WEB 2086.

The goal of the present studies was to determine whether phospholipid oxidation products and/or platelet-activating factor (PAF) are mediators of early atherogenesis in vivo. Monocyte-endothelial cell interactions have been shown to play an important role in early atherogenesis. We and others have demonstrated that PAF and phospholipid oxidation products, present in atherosclerotic lesions, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-epoxyisoprostane E(2)-sn-glycero-3-phosphocholine (PEIPC), mediate the activation of monocytes and/or endothelial cells in vitro. Previous studies have shown that the action of PAF and PAF-like ether-containing phospholipids was inhibited by WEB 2086. We now demonstrate that pretreatment of human aortic endothelial cells with WEB 2086 (10 micromol/L) and several other PAF antagonists before treatment with POVPC and PEIPC but not PGPC prevented the activation of the endothelial cells to bind monocytes. We present evidence to suggest that this inhibition is not mediated by the PAF receptor. The role of bioactive oxidized phospholipids in fatty streak formation was tested using C57BL/6J LDL R-/- mice fed a chow or Western diet for 5 weeks with or without WEB 2086 mixed with drinking water. Quantitative electrospray ionization mass spectrometry showed similar concentrations of WEB 2086 in the plasma of mice on both diets ( approximately 4 to 5 micromol/L); this concentration was inhibitory in vitro. Administration of WEB 2086 did not affect the lipid composition of mouse plasma. However, fatty streak formation was reduced by 62% in animals fed a Western diet, whereas no change was observed in the small lesions of mice on a chow diet. These studies provide evidence that PAF and/or PAF-like phospholipid oxidation products are important mediators of atherosclerotic lesion development in vivo and that specific receptor antagonists for these molecules may represent a novel therapeutic modality.

Animals↗
PubMed1999

Blocking very late antigen-4 integrin decreases leukocyte entry and fatty streak formation in mice fed an atherogenic diet.

Atherosclerotic lesion development is characterized by the recruitment of leukocytes, principally monocytes, to the vessel wall. Considerable interest has been focused on the adhesion molecule(s) involved in leukocyte/endothelial interactions. The goal of the present study was to determine the role of the very late antigen-4 (VLA-4) integrin/ligand interaction in fatty streak development using murine models. Because alpha4 null mice are not viable, a peptidomimetic was used to block VLA-4-mediated leukocyte binding. The ability of a synthetic peptidomimetic of connecting segment-1 (CS-1 peptide) to block the recruitment of leukocytes and the accumulation of lipid in the aortic sinus of either wild-type mice (strain C57BL/6J) or mice with a low-density lipoprotein null mutation (LDLR-/-) maintained on an atherogenic diet was assessed. The active (Ac) CS-1 peptide or scrambled (Sc) CS-1 peptide was delivered subcutaneously into mice using a mini osmotic pump. Mice were exposed to the peptide for 24 to 36 hours before the onset of the atherogenic diet. In C57BL/6J mice, leukocyte entry into the aortic sinus, as assessed by en face preparations, was inhibited by the active peptide (Ac=28+/-4, Sc=54+/-6 monocytes/valve; P=0.004). Additionally, frozen sections stained with Oil Red O were analyzed to assess lipid accumulation in the aortic sinus. C57BL/6J mice that received the (Ac) compound demonstrated significantly reduced lesion areas as compared with mice that received the (Sc) peptide (Ac=4887+/-4438 microm2, Sc=15 009 +/-5619 microm2; P<0.0001). In a separate study, LDLR-/- mice were implanted with pumps containing either the (Ac) or (Sc) peptide before initiation of the atherogenic diet. Because LDLR-/- mice fed a chow diet displayed small lesions at 14 weeks, the effects of the peptide seen in these animals represented a change in early lipid accumulation rather than initiation. By using whole-mount preparations, the (Ac) but not the (Sc) peptide significantly reduced the area of lipid accumulation in the aortic sinus, resulting in an approximate 66% decrease. Plasma analysis from all studies revealed concentrations of peptide to be present at levels previously determined by in vitro analysis to block adhesion. (Ac) CS-1 peptide, which blocks VLA-4 on the leukocyte surface, is effective in reducing leukocyte recruitment and lipid accumulation in the aortic sinus. The present study provides in vivo evidence that the VLA-4 integrin plays an important role in the initiation of the atherosclerotic lesion and lipid accumulation, and it suggests a potential therapeutic strategy for this disease.

Animals↗
PubMed1999

High-density lipoprotein increases intracellular calcium levels by releasing calcium from internal stores in human endothelial cells.

Elevated levels of high-density lipoproteins (HDL) appear to delay or prevent the development of atherosclerosis. The intracellular signaling mechanisms activated by HDL in vascular cells are currently under active investigation. In this study the effects of HDL on endothelial intracellular Ca levels (EC Ca(i)) are investigated. We show that HDL, like low density lipoproteins (LDL), increases EC Ca(i) in a dose-dependent fashion by releasing Ca from internal stores. Neither apolipoprotein A-I (apo A-I) nor apolipoprotein A-II (apo A-II) was responsible for the increase in EC Ca(i). HDL appeared to release Ca from the same internal stores as did LDL, since preincubation of EC with LDL prevented subsequent responses to HDL but not to the vasodilator ATP. In addition, preincubation of EC with pertussis toxin, an inhibitor of specific G proteins, as well as U73122, an inhibitor of phospholipase C, prevented a rise in EC Ca(i) in response to HDL. These findings suggest that HDL, like LDL, can modulate EC Ca(i) and that this occurs via a pertussis toxin-sensitive G protein-mediated pathway which involves phospholipase C.

Adenosine Triphosphate↗
PubMed1999

A comparison of the early inflammatory effects of an agr-/sar- versus a wild type strain of Staphylococcus aureus in a rat model of endophthalmitis.

PURPOSE: We examined the ability of a wild type and an isogenic mutant strain of Staphylococcus aureus, deficient in the production of hemolysins and lipase (agr (-)/sar (-)), to induce endophthalmitis and inflammatory cell infiltration into the eye at 6, 24 and 48 hours after injection in a rat model of endophthalmitis. METHODS: Rat eyes were injected with 25 microl of viable S. aureus or sterile saline. Eyes were graded for clinical signs of inflammation daily, removed and processed for standard histologic analysis 6, 24 and 48 hours after injections. Comparisons of clinical scores and mean inflammatory cell numbers were made between S. aureus and control injected eyes. RESULTS: Both experimental groups developed clinical signs of endophthalmitis and demonstrated infiltration of inflammatory cells at 24 and 48 hours. Clinical inflammation in the Mutant I group was less than the wild type group at these times and significantly less at 48 hours (p<0.05). No statistically significant difference in the number of inflammatory cells was detected between the wild type and Mutant I injected eyes at 24 hours. At 48 hours, inflammatory cells increased by 75.0% in the wild type group and decreased by 19.0% in the Mutant I group and a statistically significant difference was seen between these two groups (p<0.05). At all times, the majority of inflammatory cells were neutrophils. By 48 hours, an increase in monocytes-macrophages was noted. CONCLUSION: Both strains of S. aureus induced clinical signs of inflammation and inflammatory cell infiltration. Clinical inflammation and inflammatory cell numbers were less in rats injected with the Mutant I strain. These results suggest that hemolysins and lipase may be important in the early induction phase of the inflammatory response.

Animals↗