Abstract-Atherosclerosis is a disease of the arterial wall that seems to be tightly modulated by the local inflammatory balance. Whereas a large body of evidence supports a role for proinflammatory mediators in disease progression, the understanding of the role of the antiinflammatory component in the modulation of plaque progression is only at its beginning. TGF-␤1, -␤2, and -␤3 are cytokines/growth factors with broad activities on cells and tissues in the cardiovascular system and have been proposed to play a role in the pathogenesis of atherosclerosis. However, no study has examined the direct role of TGF-␤ in the development and composition of advanced atherosclerotic lesions. In the present study, we show that inhibition of TGF-␤ signaling using a neutralizing anti-TGF-␤1, -␤2, and -␤3 antibody accelerates the development of atherosclerotic lesions in apoE-deficient mice. Moreover, inhibition of TGF-␤ signaling favors the development of lesions with increased inflammatory component and decreased collagen content. These results identify a major protective role for TGF-␤ in atherosclerosis.

IntroductionA role for von Willebrand factor (VWF) as a chaperone molecule for procoagulant factor VIII (FVIII) has been extensively documented. [1][2][3][4] Under physiologic conditions, VWF binds to FVIII after its release in the circulation. VWF protects FVIII from proteolysis by lipid-bound proteases, stabilizes the FVIII heterodimeric structure, modulates its activity by thrombin, and further regulates its elimination by lipoprotein-related receptors. 5,6 Patients with severe hemophilia A lack functional endogenous FVIII. In up to 30% of the patients, injection of exogenous FVIII to treat hemorrhages results in the development of anti-FVIII antibodies that inhibit the therapeutically administered FVIII. In vivo experimental evidence and clinical observations suggest that the presence of VWF in FVIII preparations is associated with reduced FVIII immunogenicity. 7,8 Cellular and molecular mechanisms underlying a putative protective effect of VWF remain, however, unclear.The induction of a primary anti-FVIII immune response requires the administered FVIII to be endocytosed by professional antigen-presenting cells (APCs) and to be presented to FVIIIspecific naive CD4 ϩ T lymphocytes. In previously untreated patients, dendritic cells (DCs) are presumably the only candidate professional APCs. We hypothesized that VWF protects FVIII from being endocytosed by DCs, thus leading to reduced antigen presentation and stimulation of immune effectors. Materials and methodsDCs were generated from circulating monocytes of healthy blood donors on 5-day culture in X-VIVO 15 -1% AB serum or in RPMI-10% FCS, supplemented with rhGM-CSF (1000 UI/10 6 cells; Immunotools, Friesoythe, Germany) and rhIL-4 (500 UI/10 6 cells; R&D Systems, Lille, France). Surface phenotypic expression confirmed their immature status (data not shown).DCs generated in X-VIVO 15 -1% AB serum were incubated with FVIII-FITC (molar ratio, 1:25) alone or in the presence of VWF (Wilfactin) or human serum albumin (HSA). Conjugation of FVIII with FITC did not alter its specific activity (Ͼ 4000 IU/mg) and its interaction with a series of monoclonal anti-FVIII IgG (data not shown) and with VWF ( Figures S1 and S4, available on the Blood website; see the Supplemental Figures link at the top of the online article).DCs from donors with the DRB1*1501/DRB5*01 haplotype, generated in RPMI-10% FCS, were used for T-lymphocyte activation studies. The synthetic FVIII-derived I 2144 -T 2161 peptide (NeoMPS, Strasbourg, France) and human recombinant factor IX (Benefix) were used as controls.For confocal microscopy studies, DCs were fixed with 100% ethanol and mounted on glass slides. Images were acquired using a Leica SP2 confocal microscope (Leica, Mannheim, Germany) coupled to a Leica DMIRE2 inverted microscope (Wetzlar, Germany). The detection wavelength range was 500 to 580 nm for FITC. Results and discussionWe first analyzed the kinetics of internalization of FITC-labeled FVIII by DCs. Incubation of DCs with FVIII-FITC resulted in a dose-dependent labeling of the cells ...