370 Loss of Phosphoinositide 3-KINASE Gamma Activity Leads to Decreased Neointima Formation After Vascular Injury in Mice (original) (raw)
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Complement C5a inhibition reduces atherosclerosis in ApoE –/– mice
The FASEB Journal, 2011
The complement C5a receptor, CD88, is present on many of the cells found within human atherosclerotic plaques, but little is known about the role of C5a in atherogenesis. Using real-time PCR, we determined that ApoE ؊/؊ mice fed a normal diet express more aortic CD88 mRNA compared with controls, and this increase coincides with atherosclerotic lesion development (P<0.001 for 3-vs. 25-wk-old animals). Conversely, mRNA expression of the alternative C5a receptor, C5L2, in aortas of ApoE ؊/؊ mice, was lower than controls at all time points. Using immunohistochemistry, we confirmed the presence of CD88 on macrophages, smooth muscle cells, and activated endothelial cells in plaques from brachiocephalic arteries. Treatment of ApoE ؊/؊ mice with a CD88 antagonist (PMX53; 3 mg/kg s.c. 3؋/wk plus 1 mg/kg/d p.o.) for 25 wk reduced lesion size and lipid content in the plaque by ϳ40% (P<0.05). Our study provides evidence for a proatherogenic role for C5a and identifies the CD88 antagonist PMX53 as a potential antiatherosclerotic drug.
The American Journal of Pathology, 2014
The complement anaphylatoxin C5a functions through its two receptors, C5aR (CD88) and C5a receptorlike 2 (C5L2). Their role in atherosclerosis is incompletely understood. We, therefore, analyzed C5aR and probed the yet unknown expression and function of C5L2 in human atherogenesis. Human atherosclerotic plaques obtained by endarterectomy were staged and analyzed for C5L2 and C5aR by IHC and quantitative real-time PCR. C5L2-expressing cells in plaques were mostly macrophages, less neutrophils and endothelial cells, as determined by double immunostaining. Although early influx of C5aR þ cells was detected, C5L2 levels increased with lesion complexity and colocalized with C5aR and oxidized lowdensity lipoprotein. Gene expression of C5L2 and C5aR showed similar trends, such as the receptorexpressing cells. The expression of C5L2 in advanced lesions correlated with increased levels of IL-1b and tumor necrosis factor-a in plaques. Furthermore, in vitro experiments in macrophages from wildtype and C5l2-and C5ar-deficient mice corroborated the contributing role of C5l2 in oxidized lowdensity lipoproteinepretreated C5a-induced cytokine expression, as measured by enzyme-linked immunosorbent assay. Finally, C5l2-and C5ar-deficient peripheral blood mononuclear cells showed less arrest on tumor necrosis factor-aestimulated mouse endothelial cells in vitro when compared with wild-type controls. Taken together, prominent C5L2 expression in advanced atherosclerotic stages directly correlates with high levels of proinflammatory cytokines. This might indicate a proinflammatory role of C5L2 in atherosclerosis that needs to be pursued in the future by applying in vivo mouse models. (Am J Pathol 2014, -: 1e11; http://dx.
Expression and Function of C5a Receptor in Mouse Microvascular Endothelial Cells
The Journal of Immunology, 2002
The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K d50 of 3.6 nM and to ϳ15,000 -20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [ 125 I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-␥, or IL-6 in a time-and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured.
C5a promotes migration, proliferation, and vessel formation in endothelial cells
Inflammation Research, 2010
Objectives The goal of this paper is to investigate the effects of activated complement C5a on vascular endothelium during vessel formation. Methods A human microvascular endothelial cell line (HMEC-1) derived from post-capillary venules in skin was used to measure DNA synthesis, proliferation and cellcycle progression. In vitro ring-shaped formation by the cells was assessed by using type I collagen gel matrix and a cell-migration assay using the Chemotaxicell chamber. A Matrigel plug assay was performed to confirm the effect of C5a in vivo. Results C5a progressed the cell cycle of HMEC-1 into G2/M phases, and induced DNA synthesis and proliferation in a dose-dependent manner. C5a efficiently induced migration and ring-shaped structure formation both in vitro and in vivo. Furthermore, a C5a receptor antagonist (W-54011) suppressed all HMEC-1 activities including proliferation and migration.
Circulation, 2006
Background-Venous bypass grafts may fail because of development of intimal hyperplasia and accelerated atherosclerosis. Inflammation plays a major role in these processes. Complement is an important part of the immune system and participates in the regulation of inflammation. The exact role of complement in the process of accelerated atherosclerosis of vein grafts has not yet been explored, however. Methods and Results-To assess the role of complement in the development of vein graft atherosclerosis, a mouse model, in which a venous interposition was placed in the common carotid artery, was used. In this model, vein graft thickening appeared within 4 weeks. The expression of complement components was studied with the use of immunohistochemistry on sections of the thickened vein graft. C1q, C3, C9, and the regulatory proteins CD59 and complement receptor-related gene y could be detected in the lesions 4 weeks after surgery. Quantitative mRNA analysis for C1q, C3, CD59, and complement receptor-related gene y revealed expression of these molecules in the thickened vein graft, whereas C9 did not show local mRNA expression. Furthermore, interference with C3 activation with complement receptor-related gene y-Ig was associated with reduced vein graft thickening, reduced C3 and C9 deposition, and reduced inflammation as assessed by analysis of influx of inflammatory cells, such as leukocytes, T cells, and monocytes. In addition, changes in apoptosis and proliferation were observed. When C3 was inhibited by cobra venom factor, a similar reduction in vein graft thickening was observed. Conclusions-The complement cascade is involved in vein graft thickening and may be a target for therapy in vein graft failure disease.
Complement C3a and C5a Induce Different Signal Transduction Cascades in Endothelial Cells
The Journal of Immunology, 2002
In leukocytes, C3a and C5a cause chemotaxis in a G i -dependent, pertussis toxin (PT)-sensitive fashion. Because we found that HUVECs and immortalized human dermal microvascular endothelial cells express small numbers of C3aRs and C5aRs, we asked what the function of these receptors was on these cells. Activation of the C3aR caused transient formation of actin stress fibers, which was not PT-sensitive, but depended on rho activation implying coupling to G ␣12 or G ␣13 . Activation of the C5aR caused a delayed and sustained cytoskeletal response, which was blocked by PT, and resulted in cell retraction, increased paracellular permeability, and facilitated eosinophil transmigration. C5a, but not C3a, was chemotactic for human immortalized dermal microvascular endothelial cells. The response to C5a was blocked by inhibitors of phosphatidylinositol-3-kinase, src kinase, and of the epidermal growth factor (EGF) receptor (EGFR) as well as by neutralizing Abs against the EGFR and heparin-binding EGF-like factor. Furthermore, immune precipitations showed that the EGFR was phosphorylated following stimulation with C5a. The C5aR in endothelial cells thus uses a signaling cascade-transactivation of the EGFR-that does not exist in leukocytes, while the C3aR couples to a different G protein, presumably G ␣12/13 .
Arteriosclerosis, Thrombosis, and Vascular Biology, 2012
Objective—The goal of this study was to assess whether immunization ofLdlrtm1HerApobtm2SgyJ mice with 2 peptides located at the N-terminus of the C5a receptor (C5aR), either alone or in combination, is effective in reducing atherosclerotic lesions.Methods and Results—Five- to 6-week-old femaleLdlrtm1HerApobtm2SgyJ mice were immunized using a repetitive immunization multiple sites strategy with keyhole limpet hemocyanin-conjugated peptides derived from the C5aR, either alone (designated as C5aR-P1 [aa 1–21] and C5aR-P2 [aa 19–31]) or in combination (designated as C5aR-P1+C5aR-P2). Mice were fed a high-fat diet for 10 weeks. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants. Immunization ofLdlrtm1HerApobtm2SgyJ mice with these peptides elicited high concentrations of antibodies against each peptide. Immunization with the single peptide...
C5a-Induced Gene Expression in Human Umbilical Vein Endothelial Cells
American Journal of Pathology, 2004
The endothelium plays a critical role in the inflammatory process. The complement activation product, C5a, is known to have proinflammatory effects on the endothelium, but the molecular mechanisms remain unclear. We have used cDNA microarray analysis to assess gene expression in human umbilical vein endothelial cells (HUVECs) that were stimulated with human C5a in vitro. Chip analyses were confirmed by reverse transcriptase-polymerase chain reaction and by Western blot analysis. Gene activation responses were remarkably similar to gene expression patterns of HUVECs stimulated with human tumor necrosis factor-␣ or bacterial lipopolysaccharide. HUVECs stimulated with C5a showed progressive increases in gene expression for cell adhesion molecules (eg, E-selectin, ICAM-1, VCAM-1), cytokines/chemokines, and related receptors (eg, VEGFC, IL-6, IL-18R). Surprisingly, HUVECs showed little evidence for up-regulation of complement-related genes. There were transient increases in gene expression associated with broad functional activities. The three agonists used also caused down-regulation of genes that regulate angiogenesis and drug metabolism. With a single exception, C5a caused little evidence of activation of complement-related genes. These studies indicate that endothelial cells respond robustly to C5a by activation of genes related to progressive expression of cell adherence molecules, and cytokines and chemokines in a manner similar to responses induced by tumor necrosis factor-␣ and lipopolysaccharide.