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CLINICAL RESEARCH: ATHEROSCLEROSIS

CD40 Ligand+ Microparticles From Human Atherosclerotic Plaques Stimulate Endothelial Proliferation and Angiogenesis

A Potential Mechanism for Intraplaque Neovascularization

Aurélie S. Leroyer, PhD^_*,, Pierre-Emmanuel Rautou, MD^_*,, Jean-Sébastien Silvestre, PhD^_*,, Yves Castier, MD, PhD, Guy Lesèche, MD, PhD, Cécile Devue, BSc^_*,, Micheline Duriez, BSc^_*,, Ralf P. Brandes, MD, PhD, Esther Lutgens, MD, PhD^||, Alain Tedgui, PhD^_*, and Chantal M. Boulanger, PhD^_*,,_*

^_* Institut National de la Santé et de la Recherche Médicale, Cardiovascular Research Center INSERM Lariboisière, Paris
Paris VII University, Paris
Assistance Publique Hopitaux de Paris, AP-HP Hopital Bichat, Vascular Surgery Department, Paris, France
Institut fur Kardiovasculare Physiologie, Johann Wolfgang Goethe-Universitat, Frankfurt-am-Main, Germany
^|| Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, the Netherlands

Manuscript received May 5, 2008; revised manuscript received July 3, 2008, accepted July 28, 2008.

^_* Reprint requests and correspondence: Dr. Chantal M. Boulanger, INSERM CRC Lariboisière, Unité 689, 41 bd Chapelle, 75475 Paris, France (Email: chantal.boulanger{at}inserm.fr).

Objectives: Our goal was to demonstrate that microparticles (MPs) are the endogenous signal leading to neovessel formation through CD40 ligation in human atherosclerotic plaques.

Background: Vulnerable atherosclerotic plaques prone to rupture are characterized by an increased number of vasa vasorum and frequent intraplaque hemorrhage. Although inflammatory cytokines, growth factors, or CD40/CD40 ligand (CD40L) are possible candidates, the mechanism of atherosclerotic plaque neovascularization remains unknown. Atherosclerotic plaques contain large amounts of membrane-shed submicron MPs released after cell activation or apoptosis.

Methods: Microparticles were isolated from endarterectomy specimens surgically obtained from 26 patients and characterized by phosphatidylserine exposure and specific markers of cellular origin.

Results: Plaque MPs increased both endothelial proliferation assessed by ³H-thymidine incorporation and cell number and stimulated in vivo angiogenesis in Matrigel (BD Biosciences, San Diego, California) assays performed in wild-type and BalbC/Nude mice, whereas circulating MPs had no effect. Microparticles from symptomatic patients expressed more CD40L and were more potent in inducing endothelial proliferation, when compared with asymptomatic plaque MPs. Most of CD40L+ MPs (93%) isolated from human plaques were of macrophage origin. Microparticle-induced endothelial proliferation was impaired by CD40L or CD40-neutralizing antibodies and abolished after endothelial CD40-ribonucleic acid silencing. In addition, the proangiogenic effect of plaque MPs was abolished in Matrigel assays performed in the presence of CD40L-neutralizing antibodies or in CD40-deficient mice.

Conclusions: These results demonstrate that MPs isolated from human atherosclerotic lesions express CD40L, stimulate endothelial cell proliferation after CD40 ligation, and promote in vivo angiogenesis. Therefore, MPs could represent a major determinant of intraplaque neovascularization and plaque vulnerability.

Key Words: atherosclerosis • microparticles • inflammation • angiogenesis • proliferation • VEGF

Abbreviations and Acronyms   AnnV = Annexin V   CD40L = CD40 ligand   COX = cyclooxygenase   DMEM = Dulbecco's modified eagle medium   ELISA = enzyme-linked immunoadsorbent assay   FCS = fetal calf serum   FITC = fluoroisothiocyanate   HUVEC = human umbilical vein endothelial cell   IL = interleukin   MP = microparticle   PC5 = phycoerythrin-cyanin 5   PE = phycoerythrin   siRNA = small interfering ribonucleic acid   VEGF = vascular endothelial growth factor

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