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CD40 Ligand Microparticles From Human Atherosclerotic Plaques Stimulate Endothelial Proliferation and Angiogenesis JACC Vol. 52, No. 16, 2008 - A Potential Mechanism for Intraplaque Neovascularization -
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Background Atherosclerosis - inflammatory disease of the vascular wall - lipid accumulation → macrophage infiltration → focal thickening of the intimal layer of arteries → lesion ruptures → local thrombus formation → arterial occlusion → tissue ischemia Vulnerable atherosclerotic plaques - enlarged necrotic core containing apoptotic macrophages - increased number of vasa vasorum - more frequent intraplaque hemorrhage leaky neovessels → in traplaque hemorrhage → erythrocyte-derived free cholesterol within the lipid core / excessive macrophage Infiltration : stable → unstable plaque
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Background mechanism of intraplaque neovascularization ? → Ligation of CD40? - CD40 and CD40 ligand(CD40L) are highly expressed in human atherosclerotic plaques - CD40 ligation → expression of several angiogenic factors - interruption of CD40 signaling → plaque stability ↑ Advanced human atherosclerotic plaques contain large amounts of microparticles (MPs) MPs : submicron membrane vesicles released after cell activation or apoptosis Microparticles from human atherosclerotic lesions - thrombogenic - from macrophages, lymphocytes, erythrocytes, and smooth muscle, endothelial cells - inflammatory and angiogenic responses(in vitro) hypothesis : MPs are the endogenous signal leading to neovessel formation through ligation of CD40 in human atherosclerotic plaques.
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Methods MP isolation from human endarterectomy specimens. 26 patients : carotid endarterectomy surgical samples : rinsed in cold sterile phosphate-buffered saline solution supplemented with 100 U/ml streptomycin and 100 U/ml penicillin atherosclerotic lesions from healthy vessel wall. MPs : minced using fine scissors in fresh Dulbecco’s modified eagle medium (DMEM) → centrifuged first at 400 g (15min) plaque homogenate : 12,500 g (5 min) supernatants pellet MPs : further centrifuged at 20,500 g for 150 min at 4°C vehicle : DMEM alone
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Methods Circulating MP isolation from platelet-free plasma obtained from the same patients citrated venous blood (5 ml) → centrifuged at 500 g for 15 min → at 13,000 g for 5 min at room temperature → dilution in endothelial cell medium → centrifugation at 20,500 g for 150 min (4°C)
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Methods Flow cytometry analysis plaque homogenate and platelet-free plasma incubated 10 ul of plaque homogenate room temperature for 30 min in the dark. - with different fluorochrome-labeled antibodies - with their corresponding isotype-matched IgG control specimens different fluorochrome-labeled antibodies : Anti-CD154(CD40L)-PE, anti–CD4-PE, anti–CD41-PC5, anti–62P FITC, anti– CD66b-FITC, anti–CD144-PE, and anti–CD235a-FITC, antihuman CD14-PE conjugated antihuman CD154-FITC or isotype for colabeling with PE-conjugated antibodies : To determine the cellular origin of CD40L MPs FITC-conjugated Annexin V (AnnV) : presence of phosphatidylserine at the surface of plaque MPs :
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Methods Assessment of cell proliferation Endothelial cell proliferation was determined by 3H-thymidine incorporation. Human umbilical vein endothelial cells (HUVECs) stimulated with either MPs, MPs’ vehicle, or FCS during 48 h. Enzyme-linked immunoadsorbent assay (ELISA). IL-8 release quantification CD40L quantification. VEGF release quantification
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Methods RNA-mediated silencing of CD40 in HUVECs. RNA interference treatment Transfection of small interfering RNA(siRNA) concentration Transfection efficacy was assessed by flow cytometry analysis of CD40 epression on HUVECs after 72 h.
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Methods Matrigel assay 10-week-old BalbC/Nude or C57Bl/6 and CD40/-deficient female mice → 0.5-ml SC inj. of Matrigel containing either 3X10 6 plaque MPs → Matrigel rapidly formed a subcutaneous plug that was left for 7 days. → mice were euthanized, and the skin of the mouse was easily pulled back to expose the Matrigel. → Plugs were removed → fixed with 4% formaldehyde at 4°C for 12 h → embedded in paraffin, sectioned, → stained with Masson Trichrome Neovessel formation -score 0 : absence of cells in Matrigel section -score 1 : presence of endothelial cells -score 2 : presence of erythrocytes into the Matrigel plug. endothelial cells - antimouse CD31-FITC or FITC-labeled GSL-1-isolectin B4 erythrocytes - antimouse PE-labeled TER-119 antibody
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Methods Statistical analysis. Wilcoxon tests : nonparametric paired samples Mann-Whitney U tests : independent samples Differences were considered significant with a value of p <0.05.
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Results Plaque MPs induce in vitro endothelial cell proliferation
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Results Mechanism of plaque MP-induced endothelial cell proliferation
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Results Plaque MPs induced in vivo angiogenesis.
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Discussion Shed-membrane MPs isolated from human atherosclerotic lesions stimulate endothelial cell proliferation and promote in vivo neovessel formation after CD40 ligation Symptomatic patients : CD40L + MPs ↑ Accumulation of MPs in atherosclerotic lesions : endogenous signal for atherosclerotic plaque neovascularization and vulnerability. Shed-membrane vesicles from human atherosclerotic plaque - from cell plasma membrane budding after inflammation of the vessel wall - activation or apoptosis of cells from the vascular wall, as indicated by their multiple cellular origins Accumulation of oxidized lipids and apoptotic cells in atherosclerotic lesions → MP phagocytosis and removal by macrophages↓ → thrombogenic method to isolate MPs from human atherosclerotic lesions - not generate MPs from healthy human arteries
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Discussion shed-membrane MPs can disseminate information to neighboring or remote cells -MPs of different cellular origin stimulate proinflammatory responses in endothelial cells -endothelial-, platelet-, and tumor-cell derived MPs are capable of either promoting or inhibiting angiogenesis through reactive oxygen species, metalloproteinases, and growth factors such as VEGF or sphingomyelin(in vitro generated MPs) MP composition (both lipid and protein fractions) vary depending on the stimulus initiating their Vesiculation MPs isolated from human atherosclerotic plaques → endothelial cell proliferation MPs isolated from the blood → no endothelial proliferation (in vivo) symptomatic plaques > asymptomatic plaques. in atherosclerotic lesions, MPs could determine neovascularization and may favor plaque instability.
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Discussion angiogenesis is regulated by the combined action of various cytokines and growth factors released by infiltrating inflammatory cells → mechanism? → CD40 ligation? Both CD40 and CD40L are highly expressed in human atherosclerotic plaques CD40 ligation stimulates endothelial proliferation and angiogenesis Blockade of CD40 signaling improves plaque stability(in vivo) CD40L is bound to macrophage-derived MPs isolated from human atherosclerotic plaques CD40L interaction with endothelial CD40 → proliferative effect of MPs isolated from human plaque. in vivo neovessel formation induced by plaque MPs requires the presence of functional CD40 in mice → pathways? → VEGF and the PI3-kinase/Akt
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Conclusions MPs = potential endogenous triggers of neovascularization and growth of atherosclerotic plaque. Deletery proangiogenic effect of MPs is augmented and could carry on the vicious circle of plaque instability in advanced and complex symptomatic human atherosclerotic plaque more apoptosis → more MPs → neovascularization, intraplaque hemorrhage, lesion growth → macrophage apoptosis
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