476 positive aortic endothelial cells and not medial SMC [6]. High levels of P2Y2 mRNA were detected in medial SMC 3 days after collar placement, before appearance of neo- intima. At day 14, all intimal and medial SMC were P2Y2 positive. Fura-2 digital imaging of single SMC, used to measure changes in myoplasmic calcium concentration in response to P2Y receptor agonists, confirmed an increase in P2Y2 receptor activity. However, the same study showed that P2Y4 mRNA was equivalently expressed in sham- operated and collared arteries or cultured rabbit carotid SMC whereas P2Y6 mRNA was not detected in carotid arteries or cultured SMC. In a more recent study, it was shown that P2Y2 receptor up-regulation occurs in stented porcine coronary artery, a clinically relevant model of arterial injury [54]. P2Y2 receptor mRNA levels were significantly increased in coronary SMC dispersed from stented segments of coronary arteries 3 weeks after stent angioplasty compared with SMC from unstented segments. There was no significant difference observed in levels of P2Y6 mRNA in the stented and unstented artery segments whereas P2Y4 receptor mRNA was undetectable. Up-regulation of functional P2Y receptors also occurs in the basilar artery of the rat double-hemorrhage model [7], in the coronary artery of diabetic dyslipidemic pigs [8], and in human atherosclerotic lesions (Seye and Desgranges, unpublished data). It has been proposed that up-regulation of P2Y receptors could be a potential diagnostic indicator for the early stages of atherosclerosis [9]. Interestingly, a more recent study showed that high shear stress associated with vascular diseases can selectively up-regulate P2Y2 and P2Y6 receptors in perfused arterial SMC [101]. P2X1 and P2X4 receptors have been shown to be up-regulated in rabbit intimal thickenings [102]. Taken together, these findings strongly suggest that at least some P2 receptor subtypes (most notably P2Y2) are implicated in the development of vascular disease. Pathophysiological significance of P2 receptor modulation in vascular injury Migrating and proliferating SMC in the arterial media, together with infiltrating macrophages and T lymphocytes, are the main cell types that comprise atherosclerotic and restenotic lesions [1]. In the early stages of intimal hyperplasia, SMC are modified from a differentiated, contractile phenotype to an immature, synthetic phenotype, and this enables them to migrate into the intima, proliferate, and secrete extracellular matrix components. In many respects, this shift in phenotype is a reversal of the normal differentiation pattern of vascular SMC during fetal and early postnatal life [103, 104]. However, there is still a lack of knowledge concerning the phenotypic regulation of Purinergic Signalling (2006) 2:471–480 SMC during vasculogenesis and vascular disease. A high level of P2 receptor expression could be related to the altered phenotype of SMC in intimal thickenings. Partially dedifferentiated SMC are found in rat arterial intimal lesions after balloon angioplasty [105, 106], and their phenotype has been compared with that of newborn rat aortic SMC [107]. Interestingly, it has been reported that medial SMC of rat embryonic aorta also exhibit high P2Y2 expression similar to intimal thickenings [20]. Others studies have shown that P2Y1 and P2Y2 receptor transcripts are strongly up-regulated with phenotypic changes in rat SMC whereas P2X1 mRNA is completely down-regulated and P2Y4 and P2Y6 mRNA levels are unchanged [28, 58]. Taken together, these and other results suggest that P2Y2 receptor expression is up-regulated in the entire cell population of intimal thickenings and is closely associated with a poorly differentiated phenotype of SMC. The dramatic increase in P2Y2 mRNA expression observed in balloon angioplasty-induced intimal lesions would suggest increased activity of extracellular nucleotides with conse- quent enhancement of cell proliferation and vasoreactivity. Indeed, extracellular nucleotides, particularly ATP and UTP, have been shown to induce cell-cycle progression and proliferation of cultured arterial SMC [21, 49, 50, 51] and vasoconstriction in the absence of endothelial cells [17, 108, 109]. Since both neointimal hyperplasia and vasocon- strictive remodeling have been found to be involved in postangioplasty restenosis [1, 5, 107, 110], these findings suggest that extracellular nucleotides may play a significant role in this process, at least as long as functional endothelial cells, which regulate intimal thickening [111, 112] and nucleotide-induced vasorelaxation [113, 114], are not regenerated. Increased P2Y2 receptor expression in the neointima may by itself be sufficient to enhance local effects of extracellular nucleotides on proliferation of SMC. Although expression of other P2 receptors has been described in arterial SMC [21, 29, 115], the P2Y2 receptor seems to be more specifically involved in the response of SMC to ATP and UTP [116], particularly in potentiation of proliferation [21, 50]. Effects of extracellular nucleotides are not only dependent on the nature and number of P2 receptors present on target cells but also on local concentrations of nucleotide agonists. Although in vivo concentrations of extracellular nucleotides are difficult to measure, various in vitro experiments suggest that extracellular nucleotides are released from blood and vascular cells exposed to various physicochemical conditions, e.g., stress, hypoxia, and other factors [117–119] associated with the angioplasty process. P2Y2 receptors in SMC are involved in nucleotide- induced constriction of normal arteries [17, 108, 109]. Long-lasting alterations in vasomotricity after endothelial- cell denudation, resulting in increased sensitivity to