Apoptosis of arterial smooth muscle cells (ASMCs) could play an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that extracellular adenosine induces apoptosis in various cell types. Our aim was to delineate the capacity of this nucleoside to induce ASMC apoptosis in arterial diseases. We demonstrate that adenosine dose-dependently triggers apoptosis of cultured human ASMCs. Apoptotic cell death was quantified by analysis of nuclear chromatin morphology and characterized by DNA laddering. The involvement of adenosine receptors was suggested, because neither an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, nor an inhibitor of cellular nucleoside transport, dipyridamole, was able to inhibit adenosine-induced ASMC apoptosis. In contrast, an A(1)/A(2)-adenosine receptor antagonist, xanthine amine congener, totally inhibited adenosine-induced apoptosis. Furthermore, among more selective inhibitors of P(1) purinoceptor subtypes, only alloxazine, an antagonist of A(1)- and A(2)-adenosine receptors, completely inhibited adenosine-induced ASMC apoptosis, suggesting that adenosine triggers ASMC apoptosis via either 1 or both of these receptors. However, 8-cyclopentyl-1,3-dipropylxanthine, 8-(3-chlorostyryl) caffeine, and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate, which are A(1)-, A(2a)-, and A(3)-adenosine receptor antagonists, did not inhibit adenosine-induced apoptosis, suggesting an involvement of the A(2b)-receptor in this process. Moreover, the cAMP increase followed by cAMP-dependent protein kinase activation appears essential to mediate adenosine-induced ASMC apoptosis, thus confirming the previous hypothesis. These results indicate that adenosine-induced apoptosis of ASMCs is essentially mediated via A(2b)-adenosine receptor and involves a cAMP-dependent pathway.

Key Points Maturation of vascular endothelial growth factor–induced new vessels in cornea involves a PDGF-Shh axis. Shh promotes PDGF-BB–mediated SMC migration by inducing ERK1/2 and phosphatidylinositol 3-kinase γ activation and increased motility.

Abstract-Many factors have been shown to be involved in the development of hyperplasic lesions of vessels, but the role of extracellular nucleotides remains largely unknown. The presence of P2Y and P2X nucleotide receptors on arterial endothelial and smooth muscle cells suggests a potential role for nucleotides in the vessel pathophysiology. Although the role of P2X in physiology of vessels is well documented, that of P2Y is not completely understood. We recently demonstrated that extracellular nucleotides, and particularly UTP, induced migration of cultured arterial smooth muscle cells (ASMCs). This migration is dependent on osteopontin expression and involves the Rho and mitogen-activated protein (MAP) kinase pathways. An important question is to determine the specific role of the different P2Y receptors of rat ASMCs in the UTP-induced migration process. Therefore, we first quantified mRNA levels of P2Y 2 , P2Y 4 , and P2Y 6 nucleotide receptors in cultured rat ASMCs by a competitive RT-PCR approach and demonstrated that P2Y 2 is the most highly expressed among these receptors potentially involved in the UTP-mediated response. In addition to UTP, UDP also induced ASMC migration even when UTP regeneration was inhibited, suggesting the involvement of UDP receptor P2Y 6 . Moreover, suramin, a specific antagonist of rat P2Y 2 receptor, acted as an inhibitor of UTP-induced migration. Taken together, these results suggest a prominent role for the UTP receptor, P2Y 2 , and for the UDP receptor, Key Words: purinergic receptors Ⅲ migration Ⅲ UTP Ⅲ smooth muscle cells T he important role of arterial smooth muscle cell (ASMC) migration and proliferation in arterial hyperplasia is well documented in experimental models for atherosclerosis and restenosis. 1 Although proliferation can be easily demonstrated in arterial injury models, evidence for in vivo ASMC migration is suggested only by the presence of these cells in the intima.Many factors have been shown to be involved in the development of hyperplasic lesions of vessels. 2 Among these, the role of extracellular nucleotides remains largely unknown. Two families of receptors have been identified for these compounds: inotropic P2X receptors and metabotropic P2Y receptors. The presence of P2Y and P2X receptors on endothelial and ASMCs suggests a potential role for nucleotides in the arterial pathophysiology. Although the role of P2X receptors in vasomotoricity of vessels is well documented, that of P2Y receptors in the vessel wall is still under investigation.Several studies have shown that ATP and UTP binding to P2Y G protein-coupled receptors mediates ASMC activation, 3 cell-cycle progression, 4 and cell proliferation. 5,6 Moreover, we recently demonstrated that UTP induces ASMC migration and that this migration is dependent on osteopontin expression and involves the Rho and mitogen-activated protein (MAP) kinase pathways. 7 The overexpression of the ATP/UTP P2Y 2 receptor in ASMCs of rat aortic intimal lesion 8 and in ASMCs of human coronary atherosclerotic/restenotic ...

Rationale: Blood vessel growth and patterning have been shown to be regulated by nerve-derived signals. Desert hedgehog (Dhh), one of the Hedgehog family members, is expressed by Schwann cells of peripheral nerves. Objective: The purpose of this study was to investigate the contribution of Dhh to angiogenesis in the setting of ischemia. Methods and Results: We induced hindlimb ischemia in wild-type and Dhh –/– mice. First, we found that limb perfusion is significantly impaired in the absence of Dhh. This effect is associated with a significant decrease in capillary and artery density in Dhh –/– . By using mice in which the Hedgehog signaling pathway effector Smoothened was specifically invalidated in endothelial cells, we demonstrated that Dhh does not promote angiogenesis by a direct activation of endothelial cells. On the contrary, we found that Dhh promotes peripheral nerve survival in the ischemic muscle and, by doing so, maintains the pool of nerve-derived proangiogenic factors. Consistently, we found that denervation of the leg, immediately after the onset of ischemia, severely impairs ischemia-induced angiogenesis and decreases expression of vascular endothelial growth factor A, angiopoietin 1, and neurotrophin 3 in the ischemic muscle. Conclusions: This study demonstrates the crucial roles of nerves and factors regulating nerve physiology in the setting of ischemia-induced angiogenesis.

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These data show that OPN expression in the heart induces in vivo T-cell recruitment and activation leading to chronic myocarditis, the consequence of which is myocyte destruction and hence, dilated cardiomyopathy. Thus, OPN might therefore constitute a potential therapeutic target to limit heart failure.

The yeast YLR209c (PNP1) gene encodes a protein highly similar to purine nucleoside phosphorylases. This protein specifically metabolized inosine and guanosine. Disruption of PNP1 led to inosine and guanosine excretion in the medium, thus showing that PNP1 plays an important role in the metabolism of these purine nucleosides in vivo.Purine salvage is a complex pathway allowing interconversion of bases, nucleosides, and nucleotides. In yeast, major attention has been paid to the conversion of bases into nucleotides by phosphoribosyltransferases (PRTs): adenine-PRT, hypoxanthine-guanine-PRT, and xanthine-PRT activities have been reported (19,20), and the cognate genes have been identified (1,5,6). Yeast purine nucleoside metabolism has received far less attention, and only very recently was the first yeast gene encoding a purine nucleoside metabolizing enzyme identified (11). This gene, named ADO1, encodes adenosine kinase allowing synthesis of AMP from adenosine. Although several other enzymatic activities involved in yeast purine nucleoside metabolism have been described in the past, the corresponding genes have not yet been identified. Enzymatic activities responsible for the synthesis of inosine either from adenosine by adenosine deaminase (14) or from IMP by an IMP-specific 5Ј nucleotidase have been reported (8). Also, two distinct enzymatic activities (purine nucleoside hydrolase and purine nucleoside phosphorylase [PNP]) responsible for the degradation of inosine into hypoxanthine have been reported (7). The latter two enzymes catalyze the conversion of nucleosides to bases, although through distinct enzymatic mechanisms: (i) for nucleoside hydrolase, nucleoside ϩ H 2 O3base ϩ ribose and (ii) for nucleoside phosphorylase, nucleoside ϩ P i 3base ϩ ribose-1P.As a further step toward understanding yeast purine nucleoside metabolism, we searched for open reading frames (ORFs) in the complete yeast genome sequence that would encode candidate PNP. We found an uncharacterized ORF (YLR209c) that encodes a putative polypeptide highly similar to human and bovine PNP (Fig. 1). This enzyme has been thoroughly studied, and the three-dimensional structures of the trimeric human and bovine PNPs have been solved (3, 10). Important residues for substrate binding and catalysis have been identified (4, 12), all of which (except Val263) are conserved in the yeast enzyme (shown by asterisks in Fig. 1).To gain insight into the precise function of the yeast ORF YLR209c, the protein encoded by this ORF was tagged with 10 histidine residues at its N terminus and expressed in Escherichia coli. The PNP expression plasmid was constructed as follows. The YLR209c ORF was amplified by PCR from S288c genomic DNA with the following synthetic oligonucleotides: 359 (5Ј-CGATGCTCGAGATGAGTGATATCTTGAACGT-3Ј) and 360 (5Ј-GGACCCGGGTTATAATTCCCCCATTAC GG-3Ј). The amplification product was then digested with XhoI and SmaI and inserted in the pJC20-HisN vector (15) digested with XhoI and SmaI. The BL21(DE3) E. coli strain carrying the resulting plasmi...

Abstract-Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The Ϫ96 to ϩ1 region mediated UTP-induced OPN overexpression (ϩ276Ϯ60%). Sequence analysis of this region revealed a potential site for AP-1 located at Ϫ76. When this AP-1 site was deleted, UTP-induced activation of the Ϫ96 to ϩ1 region was totally inhibited. Thus, this AP-1 (Ϫ76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation. S everal studies suggest that migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in vascular pathologies such as atherosclerosis, restenosis, and hypertension. 1 SMC migration and proliferation can be induced by many factors, including growth factors and cytokines. [2][3][4] We have previously shown that extracellular nucleotides are also able to induce cell-cycle progression of SMCs 5,6 and their migration. 7 Thus, we were interested in understanding the mechanisms by which UTP induces SMC migration. Nucleotide-induced SMC activation is mediated via G protein-coupled P2Y receptors. Their activation leads to phospholipidase C activation and consequently to [Ca 2ϩ ] i increase and protein kinase C activation. Our previous work demonstrated that P2Y 2 , P2Y 4 , and P2Y 6 receptors are expressed in cultured SMCs 8 and that P2Y 2 is overexpressed in balloon-injured rat carotids. 9 Moreover, we have shown that these receptors are involved in UTP-induced migration. 8 We also demonstrated that UTP induces the expression of the extracellular matrix protein osteopontin (OPN) 6 and that UTP-induced migration is dependent on OPN expression and binding to ␣ v ␤ 3 integrin. 7 OPN is an RGD-containing extracellular matrix (ECM) phosphoprotein involved in cell attachment 10 and migration 11,12 and prevention of apoptosis. 13 OPN expression is induced by many growth factors, hormones, and cytokines invol...