Abstract-Osteopontin (OPN) is a soluble secreted phosphoprotein that binds with high affinity to several integrins and it has been found at the site of atherosclerotic lesions. However, the role of OPN expression in vivo is still poorly understood. To investigate the physiological role of OPN in detail, we generated transgenic mice (Tg) overexpressing the OPN gene under control of the cytomegalovirus enhancer/chicken ␤-actin promoter. Key Words: aorta Ⅲ atherosclerosis Ⅲ genes Ⅲ inflammation Ⅲ smooth muscle O steopontin (OPN) is a phosphoprotein that was originally isolated from the bone. It possesses the tripeptide sequence Arg-Gly-Asp like other extracellular matrix proteins and serum proteins such as fibronectin, vitronectin, and collagen. 1,2 OPN functions as a cell adhesion and migration molecule that can bind to several ligands including ␣ v ␤ 3 integrin, CD44, and fibronectin. 3-5 A wide range of cell types including some epithelia, macrophages, T cells, and vascular smooth muscle cells (SMCs) express OPN in a constitutive or inducible fashion. 6 -9 OPN has recently emerged as a key factor in both vascular remodeling and in the development of atherosclerosis. 10 -12 In vitro studies have shown that OPN also promotes the proliferation of cultured rat vascular SMCs 13 and human coronary artery SMCs. 14 Furthermore, a previous study has shown OPN to stimulate Arg-Gly-Aspdependent endothelial migration in vitro. 15 However, the function of OPN in the vessel walls is still not yet well understood. The present study uses OPN transgenic mice (Tg) to determine the effect of OPN on SMCs in vivo and to investigate the effect of cuff-induced injury to the femoral artery in vivo. Our results show that OPN overexpression is associated with a significant increase in medial thickening with aging in vivo and in intimal thickening after arterial injury. Materials and Methods Construction of TransgeneWe prepared a transgene construct containing the cytomegalovirus enhancer/chicken ␤-actin (CAG) promoter with cDNA to murine OPN. The CAG-OPN transgene was constructed by inserting the 1.35-kb EcoRI fragment containing mouse OPN cDNA sequence into the EcoRI site of the third exon of the rabbit ␤-globin gene in pBsCAG-2. 16,17 OPN cDNA contains 34 bp of the 5Ј noncoding region, 885 bp of the coding region, and 429 bp of the 3Ј untranslated region. Sequencing revealed that the OPN cDNA was identical to the described mouse OPN cDNA. 18 The resulting plasmid was termed pCAG/OPN-9. The CAG-OPN transgene was isolated from the pCAG/OPN-9 backbone by SalI and BamHI digestion.

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Antigen (Ag) immunization induces formation of the germinal center (GC), with large, rapidly proliferating centroblasts in the dark zone, and small, nondividing centrocytes in the light zone. We identified a novel nuclear protein, GANP, that is up-regulated in centrocytes. We found that GANP was up-regulated in GC B cells of Peyer's patches in normal mice and in spleens from Ag-immunized mice. GANP-positive cells appeared in the light zone of the GC, with coexpression of the peanut agglutinin (PNA) (PNA)-positive B220-positive phenotype. The expression of GANP was strikingly correlated with GC formation because Bcl6-deficient mice did not show the up-regulation of GANP. GANP-positive cells were mostly surrounded by follicular dendritic cells. Stimulation with anti-μ and anti-CD40 induced up-regulation of ganp messenger RNA as well as GANP protein in B220-positive B cells in vitro. GANP is a 210-kd protein localized in both the cytoplasm and nuclei, with a homologous region to Map80 that is associated with MCM3, a protein essential for DNA replication. Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also up-regulated in the GC area. These results suggest that the up-regulation of GANP might participate in the development of Ag-driven B cells in GCs through its interaction with MCM3.