474 Indeed, ATP and UTP were equally effective in causing migration of SMC when ATP was prevented from degra- dation by addition of the ectonucleotidase inhibitor α,β- methylene-ATP [57]. Several P2Y receptor subtypes could be involved in nucleotide-induced migration of SMC. It has been shown in rat aortic SMC that the P2Y2 receptor is the predominant P2Y receptor subtype [28, 58] whereas lower levels of P2Y4 and P2Y1 receptor mRNA were detected [58]. The very low level of P2Y1 receptor mRNA expression was consistent with the absence of ADP- induced migration of cultured rat aortic SMC, demonstrat- ing that P2Y1 is not involved in this process [58]. In addition, the same study showed that a commercially available solution of hexokinase-treated UDP (UTP-free) induced cell migration equally as well as untreated UDP, thereby demonstrating that UDP is chemotactic for aortic SMC by activation of the P2Y6 receptor. Conversely, migration of rat aortic SMC induced by UTP occurred even when UTP degradation by nucleoside diphosphate kinase was inhibited, demonstrating the involvement of P2Y2 and/or P2Y4 receptor(s) [58]. The increased migration of SMC in response to extracel- lular nucleotides could be related to increases in extracellular matrix (ECM) protein expression. Indeed, previous studies have shown that UTP induces osteopontin expression in rat and rabbit aortic SMC [6, 51]. Increased expression of osteopontin, an RGD-containing ECM protein, is associat- ed with the activation of rat arterial SMC in vitro and in vivo [57]. The increase in osteopontin expression plays a key role in UTP-induced migration of rat aortic SMC since a monoclonal antibody against osteopontin fully abolished UTP-induced migration [57] whereas an antibody against vitronectin, another ECM protein also involved in migra- tion of human SMC [59], had no effect on the migration of rat aortic SMC [57]. UTP induces increases in osteopontin mRNA expression by increasing both osteopontin mRNA stabilization and osteopontin promoter activity [60]. Recent studies have shown that activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat osteopontin promoter is involved in UTP-induced osteo- pontin expression. Using a luciferase promoter deletion assay, Renault et al. [61] identified a new region of the rat osteopontin promoter (−1837 to −1757) that is responsive to UTP. This region contains an NFB site located at −1800 and an Ebox located at −1768. Supershift electrophoretic mobility shift and chromatin immunoprecipitation assays identified NFB and USF-1/USF-2 as DNA-binding proteins induced by UTP. Using dominant negative mutants of IB kinase and USF transcription factors, it was confirmed that NFB and USF-1/USF-2 are involved in the UTP-induced expression of osteopontin. This ability of nucleotides to act as chemoattractants for rat arterial SMC in a concentration range potentially found Purinergic Signalling (2006) 2:471–480 in pathological vessels [62] and the findings of previous studies demonstrating the mitogenic activity of extracellular nucleotides for these cells suggest that nucleotides released from mechanically stretched vascular or damaged cells during the angioplasty process may participate in arterial- wall remodeling. Role of P2 receptors in nucleotide-induced vascular inflammation In addition to their mitogenic effects, extracellular nucleo- tides may also cause cell recruitment by inducing lympho- cyte and macrophage adhesion to human pulmonary artery endothelial cells, as demonstrated in vitro [63]. Nucleotides can also modulate rat aortic SMC adhesion and migration by increasing the expression of osteopontin [51, 57], a protein involved in both processes. Moreover, extracellular nucleotides may play a role in intra-arterial attraction of monocytes by inducing an increased expression of mono- cyte-chemoattractant protein-1 by arterial SMC [21]. Stimulation of P2 receptors is coupled to the release of the proinflammatory cytokines IL-1β, IL-1α, IL-8, and TNF-α [4] that are of obvious relevance to inflammation in atherosclerosis. Activation of P2X7 receptors on mono- cytes/macrophages enhances release of proinflammatory cytokines that modulate NO production and expression of inducible NO synthase (iNOS) [64], mediators of immune cell activation that is an early step in atherosclerotic lesion development. Monocyte recruitment into the vessel wall is a complex process that includes cell rolling, firm attachment, and directed migration. It is now becoming evident that ad- hesion molecules such as VCAM-1 play an important role in leukocyte adherence to vascular endothelial cells [65, 66]. VCAM-1 expression is induced or up-regulated by proinflammatory cytokines such as TNF-α and IL-1β in cellular components of the arterial wall, including endothe- lial cells, SMC, and fibroblasts [67–69]. ATP and UTP have been shown to induce cell–cell adhesion in a human monocyte/macrophage lineage and neutrophil adherence to human endothelial cell monolayers [63, 70]. Recent studies have shown that local UTP delivery via an osmotic pump to collared rabbit carotid arteries induced intimal accumulation of macrophages, similar to oxidized low-density lipoprotein (LDL), a response that was medi- ated by activation of P2Y2 receptors [6]. Leukocyte migration depends on the activities of adhesion proteins (e.g., selectins and integrins) on leukocytes and vascular endothelial cells. We demonstrated that activation of P2Y2 receptors in endothelial cells causes expression of VCAM-1 that mediates adherence of monocytes to vascular endothelium [71], leading to their penetration into the