1. CD40 Ligand Microparticles From Human Atherosclerotic Plaques Stimulate Endothelial Proliferation and Angiogenesis JACC Vol. 52, No. 16, 2008 - A Potential Mechanism for Intraplaque Neovascularization -

2. 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

3. 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.

4. 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

5. 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)

6. 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 :

7. 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

8. 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.

9. 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

10. 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.

11. Results Plaque MPs induce in vitro endothelial cell proliferation

12. Results Mechanism of plaque MP-induced endothelial cell proliferation

15. Results Plaque MPs induced in vivo angiogenesis.

16. 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

17. 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.

18. 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

19. 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